Reshaped human antibody to human medulloblastoma cells

ABSTRACT

The present invention discloses reshaped antibody to human medulloblastoma cells comprising:  
     (A) an L chair comprising:  
     (1) a human L chain C region, and  
     (2) an L chain V region comprising human L chain FRs and L chain CDRs of mouse monoclonal antibody ONS-M21 to human medulloblastoma cells; and,  
     (B) an H chain containing:  
     (1) a human H chain C region, and  
     (2) an H chain V region comprising human H chain FRs and H chain CDRs of mouse monoclonal antibody ONS-M21 to human medulloblastoma cells.  
     Since the majority of this reshaped human antibody is derived from a human antibody and mouse CDRs have a low level of antigenicity, the reshaped human antibody of the present invention has a low level of antigenicity in humans, and is therefore expected to be useful as a therapeutic agent and diagnostic tool for brain tumors such as medulloblastoma which strongly express antigen that is recognized by this antibody.

TECHNICAL FIELD

[0001] The present invention relates to a human/mouse chimeric antibodycomprising variable regions (V regions) of a mouse monoclonal antibodyONS-M21 to human medulloblastoma cells and constant regions (C regions)of human antibody; a reshaped human antibody wherein the complementaritydetermining regions (CDRs) of human light chain (L chain) V region andhen heavy chain (H chain) V region, are substituted by the CDRs of mousemonoclonal antibody ONS-M21 to human medulloblastoma cells; and the Lchain or H chain that composes that antibody.

[0002] Moreover, the present invention provides a DNA coding for theabove-mentioned antibody, and particularly its V region. Moreover, thepresent invention relates to a vector containing the above-mentionedDNA, and particularly to an Expression vector, along with a hosttransformed with said vector. Moreover, the present invention provides aprocess for producing chimeric antibody to human medulloblastoma cellsas well as a process for producing a reshaped human antibody to humanmedulloblastoma cells.

[0003] In addition, the present invention relates to a single chain Fvcomposed by linking an H chain V region of a reshaped human antibody tohuman medulloblastoma cells and an L chain V region of said antibody. Inaddition, the present invention relates to a process for producing theabove-mentioned single chain Fv by using DNA coding for said-singlechain Fv, a recombinant vector comprising said DNA, and a hosttransformed with said recombinant vector.

BACKGROUND ART

[0004] Medulloblastoma is one type of primitive neuroectodermal tumorswhich account for 40% of all brain tumors with glioma, and is amalignant brain tumor that frequently appears in children under 10 yearsof age, and particularly between the ages of 5 to 10 years. This tumoris a typical undifferentiated tumor that mimics morphology andarrangement patterns of undifferentiated cells that compose theembryonal medullary epithelium and metrical layer. It is considered tobe an immature tumor that has the potential to differentiate into bothnerve cells and glia cells (Tamura, K. et al., Cancer Res., 49,5380-5384 (1989)). Since these types of malignant tumors exhibitsensitivity to radiation and chemotherapeutic agents, it is treated byradiotherapy, chemotherapy as well as surgery.

[0005] However, although these treatment methods alleviate symptomstemporarily, there are many cases of relapse and death within severalyears, with an average survival period being 15 months The cause of thisis believed to be that a recurrent cancer has resistance to chemotherapyand radiation.

[0006] On the other hand, accurate evaluation of brain tumors reliesheavily on histological techniques, and requires an extremely high levelof specialized knowledge as well as auxiliary diagnostic technology.

[0007] Thus, there is a pressing need for the development of diagnostictools and therapeutic drugs that enable early diagnosis and fundamentaltreatment.

[0008] Several attempts have been made at treatment through the use ofmonoclonal antibodies that recognize medulloblastoma in the past as well(refer to Kemshead, J. T. et al., Int. J. Cancer, 31, 187-195 (1983).Allan, P. M. et al., Int. J. Cancer, 31, 591-558 (1983), Jones, D. etal., Br. J. Hematol., 57, 621-631 (1984), Gross, N. et al., Cancer Res.,46, 2998-2994 (1986), Wikstrand, C. D. et al., Cancer Res., 46,5933-5940 (1986), Gibson, F. M. at al., Int. J. Cancer, 39, 554-559(1987), Feickert, H. J. et al., Cancer Res., 49, 4338-4343 (1989),Jennings, N. T. et al., J. Neurol. Sci., 89, 63-78 (1989) and Takahashi,H. et al., Neurosurg., 27, 97-102 (1990)).

[0009] However, since nearly all of these antibodies also recognizenormal tissue and other tumors, they have the disadvantage of beinginappropriate for diagnosis and treatment of medulloblastoma.

[0010] Recently, brain tumor immunotherapy has been reported that uses amonoclonal antibody of human origin that reacts to the human glioma andexhibits ADCC activity (see, Japanese Unexamined Patent Publication No.58-201994, Japanese Unexamined Patent Publication No. 59-137497 andJapanese Unexamined Patent Publication No. 4-356792).

[0011] Mouse monoclonal antibody exhibits a high degree of immunogenic(also referred to as antigenicity) in humans. For this reason, theirtherapeutic value in humans is limited. Moreover, not only do mouseantibodies inhibit anticipated effects, but they cannot be administeredfrequently without provoking an immune reaction that brings about therisk of an allergic response that presents a problem for patients.

[0012] In order to solve these problems, a process for producinghumanized antibody has been developed. Mouse antibody can be humanizedby two methods. The simpler method involves the production of chimericantibody wherein the variable region is derived from an original mousemonoclonal antibody, while the constant region is derived from asuitable human antibody. The resulting chimeric antibody contains acomplete variable region of the original mouse antibody, and can beexpected to bind antigen with the same specificity as the original mouseantibody.

[0013] Moreover, since the ratio of the protein sequence derived fromsources other then humans is essentially reduced, it is predicted tohave a low level of immunogenicity in comparison with the original mouseantibody. Although chimeric antibody effectively binds with antigen andhas a low level of immunogenicity, there is still possibility of animmune reaction to the mouse variable region (LoBuglio, A. F. et al.,Proc. Natl. Acad. Sci. USA, 86, 4220-4224, 1989).

[0014] Although more complex, the second method for humanizing mouseantibody considerably lowers the potential immunogenicity of the mouseantibody even more. In this method, complementarity determining regions(CDRs) from variable regions of a mouse antibody are transplanted intohuman antibody variable regions to produce a reshaped human antibodyvariable regions.

[0015] Next, these reshaped human antibody variable regions are linkedto human antibody constant regions. Ultimately, the portion derived fromthe non-human protein sequence of a reshaped human antibody is only theCDRs and an extremely small portion of the framework (FR). CDRs comprisehyper-variable protein sequences. These sequences do not exhibittype-specific sequences. For these reasons, a reshaped human antibodycontaining mouse CDRs ought not to have immunogenicity stronger thannaturally-occurring human antibody containing human CDRs.

[0016] The following references should be referred to with respect toreshaped human antibodies: Riechmenn, L. et al., Nature, 332, 323-327,1988; Verhoeyen M. et al., Science, 239, 1534-1536, 1988; Kettleborough,C. A. et al., Protein Engng., A, 773-783, 1991; Maeda, H. et al., HumanAntibodies and Hybridoma, 2, 124-134. 1991; Gorman S. D. et al., Proc.Natl. Acad. Sci. USA, 88, 4181-4185, 1991; Tempest P. R. et al.,Bio/Technology, a 266-271, 1991; Co, W. S. et al., Proc. Natl. Acad.Sci. USA, 88, 2869-2873, 1991; Carter, P. et al., Proc. Natl. Acad. Sci.USA, 89, 4285-4289, 1992; Co, M. S. et al., J. Immunol., 148, 1149-1154,1992; and, Sato, K. et al., Cancer Res., 53, 851-856, 1993.

[0017] As was previously stated, although it is predicted that areshaped human antibody is useful for the purpose of therapeuticaltreatment, a reshaped human antibody to human medulloblastoma cells isnot known. Moreover, there are no methods for producing a reshaped humanantibody that can be universally applied to any specific antibody. Thus,various contrivances are necessary to produce a reshaped human antibodyto a specific antigen that has sufficient activity (for example, Sato,K. et al., Cancer Res., 53, 1-6 (1993)).

[0018] The inventors of the present invention isolated and established amedulloblastoma cell line (ONS-76) from the cerebellum ofmedulloblastoma patients (Tamura, K. et al., Cancer Res., 49, 5380-5384(1989)). By then immunizing mice with said medulloblastoma cell lineONS-76, a mouse monoclonal antibody (ONS-M21) was found thatspecifically recognizes human medulloblastoma but does not cross-reactwith normal brain tissue or peripheral blood cells (Moriuchi, S. et al.,Br. J. Cancer, 68, 831-837 (1993)). Since antigen recognized by thisantibody is strongly expressed in brain tumors such as medulloblastomaand some gliders. it is anticipated to be used as a diagnostic tool aswell as directly destroy cancer cells by inducing ADCC and CDC orconjugating with toxins and radioisotopes.

DISCLOSURE OF THE INVASION

[0019] Thus, the present invention relates to a reshaped human antibodyof a mouse monoclonal antibody ONS-M21 to human medulloblastoma cells.In addition, the present invention provides a human/mouse chimericantibody useful during the course of producing said reshaped humanantibody. Moreover, the present invention relates to a geneticengineering process for producing a reshaped human antibody and achimeric antibody of mouse monoclonal antibody ONS-M21.

[0020] More specifically, the present invention relates to

[0021] (1) L chain V region of mouse monoclonal antibody ONS-M21 tohuman medulloblastoma cells and,

[0022] (2) H chain V region of mouse monoclonal antibody ONS-M21 tohuman medulloblastoma cells.

[0023] The present invention also relates to a chimeric antibody tohuman medulloblastoma cells comprising:

[0024] (1) L chain containing a human antibody L chain C region, and theabove-mentioned L chain V region of mouse monoclonal antibody ONS-M21 tohuman medulloblastoma cells; and,

[0025] (2) H chain containing human antibody H chain C region, and the Hchain V region of mouse monoclonal antibody ONS-M21 to humanmedulloblastoma cells.

[0026] The present invention moreover relates to a reshaped humanantibody of mouse monoclonal antibody ONS-M21 to human medulloblastomacells comprising:

[0027] reshaped human L chain V region comprising

[0028] (1) framework regions (FRs) of human antibody L chain V region,and

[0029] (2) CDRs of L chain V region of mouse monoclonal antibody ONS-M21to human medulloblastoma cells; and,

[0030] reshaped human H chain V region comprising

[0031] (1) FRs of human antibody H chain V region, and

[0032] (2) CDRs of H chain V region of mouse monoclonal antibody ONS-M21to human medulloblastoma cells.

[0033] In addition, the present invention relates to L chain or H chainpolypeptides that compose the various above-mentioned antibodies as wellas DNA coding for them.

[0034] Moreover, the present invention relates to expression vectorscontaining the above-mentioned DNA as well as a host transformed bythem.

[0035] In addition, the present invention relates to a process forproducing chimeric antibody to human medulloblastoma cells as well as aprocess for producing a reshaped human antibody to human medulloblastomacells.

[0036] In addition, the present invention relates to a single chain Fvregion composed by linking an H chain V region of a reshaped humanantibody to human medulloblastoma cells and an L chain V region of saidmonoclonal antibody.

[0037] Moreover, the present invention relates to DNA coding for theabove mentioned single chain Fv region. recombinant vectors that containsaid DNA and a host transformed by said recombinant vectors.

[0038] Moreover, the present invention relates to a process forproducing a single chain Fv region characterized by culturing theabove-mentioned host and recovering a single chain Fv region from saidculture.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1 indicates expression plasmids HEF-VL-gκ and HEF-VH-gγ1comprising human EP1-α promoter/enhancer useful for expression of an Lchain and H chain, respectively.

[0040]FIG. 2 is a graph indicating the binding ability of chimericantibody ONS-M21 (ChM21) to human, medulloblastoma cell line ONS-76.

[0041]FIG. 3 is a diagram of the preparation of the first version(Version “a”) of the L chain V region of reshaped human antibodyONS-M21.

[0042]FIG. 4 is a diagram of the preparation of the H chain V region ofreshaped human antibody ONS-M21.

[0043]FIG. 5 is a graph indicating the binding ability of antibodycomprising reshaped human H chain and chimeric L chain to humanmedulloblastoma call line ONS-76.

[0044]FIG. 6 in a graph comparing the binding ability of 8 types ofreshaped human ONS-M21 antibodies comprising reshaped human H chain andone of versions “a” through “h” of reshaped human L chain, to humanmedulloblastoma cell line ONS-76, with that of chimeric antibody(ChM21).

[0045]FIG. 7 is a graph comparing the binding ability of 6 types ofreshaped human ONS-M21 antibodies comprising the reshaped human H chainand one of versions “I”, “j”, “l”, “m”, “o” and “p” of reshaped human Lchain of the present invention, to human medulloblastoma cell lineONS-76, with that of chimeric antibody (ChM21) and antibodies heavingreshaped L chain versions “k” and “n”.

[0046]FIG. 8 is a schematic diagram of a preparation process of DNAcoding for the single chain Fv region of the present invention.

[0047]FIG. 9 indicates the structure of an exile of expression plasmidsused for expressing DNA coding for the single chain Fv region of thepresent invention.

[0048]FIG. 10 is a graph that indicates an antigen binding activity ofthe single chain Fv region of the present invention (scFv-hM21) incomparison with an antigen binding activity of reshaped human ONS-21antibody and the Fab fragment of said antibody using for the parameterthe inhibition of antigen binding by mouse monoclonal antibody ONS-M21.

DETAILED EXPLANATION Cloning for DNA Coding for Mouse Antibody/Region

[0049] In order to clone a DNA coding for a V region of a mousemonoclonal antibody to human medulloblastoma cells, after preparing mRNAfrom mouse monoclonal antibody-producing cells, said mRNA is convertedto double-stranded DNA using known method followed by amplifying thetarget DNA using polymerase chain reaction (PCR). It is necessary toprepare hybridoma producing a monoclonal antibody to humanmedulloblastoma cells for the supply source of mRNA One exile of thistype of hybridoma is ONS-M21. The process for producing hybridomaONS-M21 is described later in Reference Example 1.

[0050] (1) Obtaining of Whole RNA

[0051] In order to obtain whole RNA, in the present invention, afterdestroying hybridoma cells and treating with guanidine thiocyanate,cesium chloride density gradient centrifugation was performed (Chirgwin,J. M. et al., Biochemistry, 18, 5294-5299, 1979). However, methodsalready used during cloning of other protein genes can also be used,examples of which include treatment with surfactant in the presence of aribonuclease inhibitor such as vanadium compounds followed by treatmentwith phenol (Berger, S. L. et al., Biochemistry, 18, 5143-5149, 1979).

[0052] (2) Preparation of Double-Stranded cDNA

[0053] In order to obtain single-stranded DNA from whole RNA obtained inthe manner described above, single-stranded DNA complementary to wholeRNA (cDNA) can be synthesized by using the whole RNA as a template andtreating with reverse transcriptase using oligo(dT) complementary to itspolyA chain on the 3′ terminal as primer (Larrik, J. W. et al.,Bio/Technology, 7, 934-938, 1989). In addition, a random primer may alsobe used at that time.

[0054] (3) Amplification of Mouse Antibody V Region by Polymerase ChainReaction (PCR)

[0055] Next, specific amplification of mouse antibody V region isperformed from the above-mentioned cDNA using polymerase chain reaction(PCR). The primer described in Jones, S. T. et al., Bio/Technology, 9,88-89, 1991 can be used for amplification of the mouse antibody Vregion. In the determining of the primer used for cloning of mousemonoclonal antibody ONS-M21 produced by hybridoma ONS-M21, it isnecessary to type the H chain and L chain and determine the form of bothchains.

[0056] As a result of performing typing of ONS-M21 antibody using amouse monoclonal antibody isotyping kit (Amersham International Plc.),it was clear that ONS-M21 antibody has a Cκ type L chain and γ-1C type Hchain. Typing of ONS-M21 is described later in Reference Example 2.

[0057] Next, in order to amplify the kappa (κ) type L chain V region ofmouse monoclonal antibody using polymerase chain reaction (PCR), 11types of oligonucleotide primers indicated in SEQ ID Nos: 1 to 11 (MouseKappa Variable; MKV) and the oligonucleotide primer shown in SEQ ID NO:12 (Mouse Kappa Constant; MKC) are used for the 5′-terminal primer and3′-terminal primer, respectively.

[0058] The above-mentioned MKV primer hybridizes with the DNA sequencecoding for the mouse kappa type L chain leader sequence, while theabove-mentioned MKC primer hybridizes with the DNA sequence coding forthe mouse kappa type L chain C region.

[0059] In order to amplify the H chain V region of mouse monoclonalantibody, the 12 types of oligonucleotide primers indicated in SEQ IDNOs: 13 to 24 (Mouse Heavy Variable; MHV) and the oligonucleotideindicated in SEQ ID NO: 25 (Mouse Heavy Constant, MHC) are used for the5′-terminal primer and 3′-terminal primer, respectively.

[0060] Furthermore, in the present embodiment, a 5′-terminal primercontains the sequence GTCGAC that provides a site to be cleaved byrestriction enzyme SalI near the 5′-terminal, while a 3′-terminal primercontains the nucleotide sequence CCCGGG that provides a site to becleaved by restriction enzyme XmaI near the 5′-terminal. Otherrestriction enzyme cleavage sites may be used for these restrictionenzyme cleavage sites provided Whey can be used for subcloning thetarget DNA fragment coding for the variable region in a cloning vector.

[0061] Next, in order to obtain a DNA fragment coding for a targetvariable region of a mouse monoclonal antibody, after cleaving theamplification product with restriction enzymes SalI and XmaI, isolationand purification is performed using low melting temperature agarose or acolumn (PCR product purification kit (Qiagen), DNA purification kit(Geneclean II) and so forth). On the other hand, a plasmid is obtainedthat contains a DNA fragment coding for a target variable region ofmouse monoclonal antibody by cleaving a suitable cloning vector such asplasmid pUC19 with the same restriction enzymes SalI and XmaI, andligating the above-mentioned DNA fragment to this pUC19.

[0062] The cloned DNA can be sequenced using any commonly employedmethod such as an automated DNA sequencer (Applied Biosystems Inc.).

[0063] Cloning of the target DNA and determination of its sequence aredescribed in detail in Examples 1 and 2.

Complementarity Determining Region (CDR)

[0064] A pair of V regions of L chain and H chair forms an antigenbinding site. The variable regions of the L chain and H chain comprisesfour relatively well-preserved framework regions linked with threehyper-variable or complementarity determining regions (CDR) (Kabat, E.A. et al., “Sequences of Proteins of Immunological Interest”, US Dept.Health and Human Services 1983).

[0065] The majority of the portions of the above-mentioned fourframework regions (FRs) employ a β-sheet structure. As a result, threeCDRs form a loop. The CDRs may also form a portion of the β-sheetstructure in certain cases. The three CDRs are maintained at positionsthat are three-dimensionally extremely close to each other by the FRs,and contribute to the formation of an antigen binding site together withthree CDRs of the region with which it constitutes a pair.

[0066] These CDRs can be found based on the empirical rules of Kabat, E.A. et al., “Sequences of Proteins of Immunological Interest” bycomparing the amino acid sequence of the V region of the resultingantibody with known amino acid sequences of V regions of knownantibodies. A detailed explanation of this is given in Example 3.

Production of Chimeric Antibody

[0067] Prior to designing the reshaped human V region of antibody tohuman medulloblastoma cells, it is necessary to confirm that the CDRsused actually form an antigen binding region. Chimeric antibody wasproduced for this purpose. Moreover, the amino acid sequence of mouseanti-human medulloblastoma cell antibody, predicted from the nucleotidesequence of cloned DNA of monoclonal antibody ONS-M21 described inExample 1, was compared with the V regions of known mouse and humanantibodies.

[0068] Once a DNA fragment is cloned coding for the mouse L chain and Hchain v regions of monoclonal antibody ONS-M21, chimeric anti-humanmedulloblastoma cell antibody can be obtained by linking these mouse Vregions with DNA coding for human antibody constant region and thenexpressing them.

[0069] The basic method for producing chimeric antibody compriseslinking a mouse leader sequence and V region sequence in cloned cDNAwith a sequence coding for human antibody C region already present in amammalian cell expression vector.

[0070] The above-mentioned human antibody C region can be any human Lchain C region or H chain C region, examples of which include human Lchain Cκ or H chain γ-1C and γ-4C.

[0071] In order to produce chimeric antibody, two types of expressionvectors are prepared. These vectors are an expression vector comprisingDNA coding for a mouse L chain V region and a human L chain C regionunder the control by an expression control region such as anenhancer/promoter type, and an expression vector comprising DNA codingfor a mouse H chain V region and a human H chain C region under the anexpression control region such as an enhancer/promoter type. Next, hostcells such as mammalian cells are co-transformed with these expressionvectors after, which the transformed host is cultured in vitro or invivo to produce chimeric antibody (e.g. WO91-16928).

[0072] Alternatively, DNA coding for a mouse L chain V region and ahuman L chain C region, and DNA coding for a mouse L chain V region anda human H chain C region may be introduced into a single expressionvector, host cells are transformed using said vector, and thistransformed host is then cultured in vivo or in vitro to produce thedesired chimeric antibody.

[0073] A description of production of chimeric antibody is provided inExample 4.

[0074] A cDNA coding for a mouse ONS-M21 κ type L chain leader regionand a V region is subcloned using PCR and linked to an expression vectorcontaining DNA coding for human genome L chain Cκ chain region. cDNAcoding for the γ1 type H chain leader and V regions of mouse ONS-M21antibody is subcloned using PCR, and linked to an expression vectorcontaining genome DNA coding for human γ-1C region.

[0075] Using specially designed PCR primers, cDNA coding for the Vregion of mouse ONS-M21 is provided with a suitable nucleotide sequenceat its 5′-terminal and 3′-terminal to facilitate their insertion intothe expression vector as well as to ensure that they function suitablyin said expression vector (for example, transcription efficiency isimproved in the present invention by the introduction of Kozak'ssequence). Next, the V region of mouse ONS-M21, obtained byamplification by PCR, was inserted into an HEF expression vector(FIG. 1) already containing the desired human C region using theseprimers. These vectors are suitable for transient or stable expressionof genetically engineered antibodies in various mammalian cell systems.

[0076] The chimeric ONS-M21 antibody demonstrated an activity to bind tohuman medulloblastoma cells. Thus, this indicated that a correct mouse Vregion had been cloned, and that the sequence had been determined.

Design of Reshaped Human ONS-M21 Antibody V Region

[0077] In order to produce a reshaped human antibody in which the CDRsof mouse monoclonal antibody are grafted onto a human antibody, it ispreferable that a high degree of homology exists between the FRs of themouse monoclonal antibody and the FRs of the human antibody. Thus, the Vregions of the L chain and H chain of mouse ONS-M21 antibody werecompared with the V regions of all known antibodies for which structurehas been determined using the Protein Data Bank.

[0078] The L chain V region of mouse ONS-R21 most closely resembles theconsensus sequence of subgroup IV of human L chain V region (HSGIV),demonstrating homology of 61.9%.

[0079] In a comparison of the L chain V region of mouse ONS-M21 antibodywith known human antibody L chain V regions, homology of 60.4% wasdemonstrated with human L chain V region REI, a member of subgroup I ofthe human L chain V region. Thus, the FRs of REI were used as a startingmaterial for preparation of reshaped human ONS-M21 antibody L chain Vregion.

[0080] Sixteen versions of reshaped human ONS-M21 antibody L chain Vregion were designed (versions “a”-“p”). In the first version (version“a”), human FRs were identical to FR of version “a” of the L chain Vregion of reshaped human PM-1 described in WO92-19759, which is based onREI present in reshaped human CAMPATH-1H antibody (Riechmann, L. et,al., Nature, 332-327 (1988)), while mouse CDRs were identical to theCDRs in the L chain V region of mouse ONS-M21 antibody.

[0081] Tables 1 and 2 show the amino acid sequence of the L chain Vregion of mouse ONS-M21 antibody, the FRs of REI, and the L chain Vregion of the 16 versions of reshaped ONS-21 antibody. TABLE 1 Design ofReshaped Human ONS-M21 L Chain V Regions            FR1    CDR1      FR2  CDR2          1          2       3      4 5 1234567890123456789012345678901234 567890123456789 0123456 ONS-M21VL DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVA WYQQKPGQSPKPLIY SASYRYS REl DIQMTQSPSSLSASVGDRVTITCWYQQKPGKAPKLLIY RVLa DIQMTQSPSSLSASVGDRVTITC KASQNVGTNVA WYQQKPGKAPKLLIYSASYRYS RVLb ----------------------- ----------- --------------- -------RVLc ----------------------- ----------- --------------- ------- RVLd----------------------- ----------- --------------- ------- RVLe-------------------SV-- ----------- --------------- ------- RVLf-------------------SV-- ----------- --------------- ------- RVLg-------------------SV-- ----------- --------------- ------- RVLh----------------------- ----------- --------------- ------- RVLi-------------------SV-- ----------- -------QS--P--- ------- RVLJ-------------------SV-- ----------- -------QS--P--- ------- RVLk-------------------SV-- ----------- --------------- ------- RVLl-------------------SV-- ----------- -------QS--P--- ------- RVLm----------------------- ----------- -------QS--P--- ------- RVLn-------QKF------------- ----------- -------QS------ ------- RVLo----------------------- ----------- -------QS--P--- ------- RVLp----------------------- ----------- -----------P--- -------

[0082] TABLE 2 Design of Reshaped Human ONS-M21 L Chain V Regions(cont.)              FR3   CDR3     FR4   1    6         7         8  9  0 78901234567890123456789012345678 901234587 8901234567 ONS-M2IVLGVPDRFTGSGSGTDFTLTITNVQSEDLADYFC QQYNSYPRA FGGGTKLEIK RE1GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC FGQGTKVEIK RVLaGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYNSYPRA FGQGTKVEIK RVLb--------------Y----------------- --------- ---------- RVLc------------------------------F- --------- ---------- RYLd--------------Y---------------F- --------- ---------- RVLe-------------------------------- --------- ---------- RVLf------------------------------F- --------- ---------- RVLg----------------L-------------F- --------- ---------- RVLh----------------L--------------- --------- ---------- RVLi----------------L-------------F- --------- ---------- RVLj----------------L-----------D-F- --------- ---------- RVLk----------------L-----------D-F- --------- ---------- RVLl-------------------------------- --------- ---------- RVLm-------------------------------- --------- ---------- RVLn-------------------------------- --------- ---------- RVLo-------------------------------- --------- ---------- RVLp-------------------------------- --------- ----------

[0083] Note: Those underlined amino acids in the FRs of REI indicatelocations of amino acids that differ from those of the amino acidsequence of human REI (Palm, W. et al., Hoppe-Seyler's, Z. Physiol.Chem., 356, 167-191, 1975).

[0084] The H chain V region of mouse ONS-M21 most closely resembles theconsensus sequence of human H chain V region subgroup I (HSGI),exhibiting homlogy of 57.9%. In a comparison of the H chain V region ofmouse ONS-M21 antibody with known human antibody H chain V regions, itresembled extremely closely the H chain V region of human antibody Eu, amember of human H chain V region subgroup I, from FR1 to FR3(Cunningham, B. J. et al., Biochemistry, 9, 3161, 1970). Moreover, thesize of the CDRs were also extremely similar between mouse ONS-M21antibody and human antibody Eu.

[0085] Consequently, the FRs of human antibody Eu were used as astarting material for preparation of the H chain V region of a reshapedhuman ONS-M21 antibody.

[0086] However, since the amino acid sequence of the FR4 of the humanantibody Eu has a sequence that differs from the human antibody subgroupI consensus sequence, it was decided to use the amino acid sequence ofthe FR4 of human antibody ND (Kenten, J. H. et al., Proc. Natl. Acad.Sci. USA, 79, 6661-6665 (1982)), whose V region belongs to subgroup I,for the FR4 in this case.

[0087] H chain V regions of reshaped human ONS-M21 antibody weredesigned. Amino acids at positions 27, 28, 29 and 30 of human FR1 andposition 94 of FR3 were made to be identical to the amino acids of mouseONS-M21. TABLE 3 Design of Reshaped Human ONS-M21 H Chain V Region             FR1  CDR1      FR2          1         2         3     4123456789012345678901234567890 12345 67890123456789 ONS-M21VHEVQLQQSGAELVKPGASVKLSCTASGFNIK DTYIH WAKQRPEQGLEWIG EUQVQLVQSGAEVKKPGSSVKVSCKASGGTFS WVRQAPGQGLEWMG RVHaQVQLVQSGAEVKKPGSSVKVSCKASGFNIK DTYIH WVRQAPGQGLEWMG       CDR2               FR3 5          6     7         8            901223456789012345 67890123456789012222345678901234    A                 ABC ONS-M21VH RIDPADGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCAS EU RVTITADESTNTAYMELSSLRSEDTAFYFCAGRVHa RIDPADGNTKYDPKFQG RVTITADESTNTAYMELSSLRSEDTAFYFCAS   CDR3     FR4     1        1      0       1 56789012 34567890123 ONS-M21VH AYYVNQDYWGQGTSVTVSS EU ND WGQGTTVTVSS RVHa AYYVNQDY WGQGTTVTVSS

Production of Reshaped Human ONS-M21 Antibody

[0088] Production of reshaped human ONS-M21 antibody V region isdescribed in detail in Example 5.

[0089] A reshaped human ONS-M21 antibody of the present inventioncomprises:

[0090] (A) L chains lack comprising:

[0091] (1) a human L chain C region, and

[0092] (2) an L chain V region comprising human L chain FRs and L chainV region CDRs of mouse monoclonal antibody ONS-M21 to humanmedulloblastoma; and,

[0093] (B) H chains lack comprising:

[0094] (1) a human H chain C region, and

[0095] (2) an H chain V region comprising human H chain FRs and H chainV region CDRs of mouse monoclonal antibody ONS-M21 to humanmedulloblastoma.

[0096] In order to produce a reshaped human antibody that possessessufficient activity with respect to a specific antigen, it is preferableto substitute a portion of the amino acid sequence of theabove-mentioned human FRs.

[0097] In a preferable embodiment. the amino acid of position 46 of FR2of the above-mentioned L chain V region should be proline, or the aminoacids of positions 42, 43 and 46 should preferably be amino acidsderived from mouse FR such as glutamine, serine and proline, or morepreferably, should have, the amino acid sequence of RVLi, RVLj, RVLl,RVLm, RVLo or RLVp in Tables 1 and 2.

[0098] The above-mentioned human L chain C region can be any human Lchain C region, an example of which is the human κC region. Theabove-mentioned H chain C region can also be any human H chain C region,examples of which include the human γ-1C and human γ-4C regions.Alternatively, a toxin or radioisotope may be bound instead of theabove-mentioned human L chain C region and/or above-mentioned human Hchain C region.

[0099] In order to produce a reshaped human antibody, two types ofexpression vectors are prepared. These are an expression vectorcontaining DNA coding for a previously defined reshaped human L chainunder an expression control region such as an enhancer/promoter type,and another expression vector that contains DNA coding for a previouslydefined reshaped human H chain under an expression control region suchas an enhancer/promoter type. Next, host cells such as mammalian cellsare simultaneously transformed using these expression vectors afterwhich the transformed host is cultured in vitro or in vivo to produce areshaped human antibody (e.g. WO91-16927).

[0100] Alternatively, DNA coding for a reshaped L chain and DNA codingfor a reshaped human H chain may be introduced into a single expressionvector, host cells are transformed with this vector, and thistransformed host is then cultured in vivo or in vitro to produce adesired reshaped human antibody.

[0101] Moreover, Fab or Fv, or Fv-linked single-chain Fv, can beproduced in a suitable host and used for the purpose described above(refer, for example, to Bird, et al., TIBTECH, 9, 132-137 (1991)).

[0102] Chimeric antibody or humanized antibody produced by culturing atransformed host transformed with a gene coding for the desired chimericantibody or humanized antibody in the manner described above can then beisolated from inside or outside cells and purified to homogeneity.

[0103] Furthermore, separation and purification of the chimeric antibodyor humanized antibody, which is the target protein of the presentinvention, can be performed using a protein A agarose column. Inaddition, other isolation and purification methods commonly used withproteins may also be used, and there are no limitations whatsoever onthose methods. For example, chimeric antibody or humanized antibody canbe isolated and purified by suitably selecting and combining varioustypes of chromatography, ultrafiltration, salting, dialysis and soforth.

[0104] Any expression system can be used for producing a chimericantibody or a reshaped human antibody to human medulloblastoma cells ofthe present invention, examples of which include eucaryotic cells suchas animal cells including established mammalian cell systems, mold cellsand yeast calls as well as procaryotic cells such as bacterial cellsincluding Escherichia coli cells. A chimeric antibody or a reshapedantibody of the present invention is preferably expressed in mammaliancells such as COS cells or CHO cells.

[0105] In these cases, useful, conventional used promoters can be usedfor expression in mammalian cells. For example, the use of humancytomegalovirus immediate early (HCMV) promoter is preferable. Examplesof expression vectors containing HCMV promoter include HCMV-V_(H)-HCγ1and HCMV-V_(L)-HCκ derived from pSV2neo (refer to InternationalUnexamined Application WO92-19759).

[0106] In addition, promoters derived from mammalian cells such aspromoters of viruses including retrovirus, polio virus, adenovirus andsimian virus 40 (SV40) as well as human polypeptide chain elongationfactor1α (HEF-1α) should be used for promoters of genetic expression inmammalian cells that can be used for the present invention. For example,in the case of using SV40 promoter, expression can be easily carried outby following the method Mulligan, R. C. et al. (Nature, 277, 108-114(1979)), or in the case of using HEF1α promoter, the method ofMizushima, S. et al. (Nucleic Acids Research, 11, 5322 (1990)).

[0107] Those derived from SV40. polio virus, adenovirus, bovinepapilloma virus (BPV) and so forth can be used for an origin ofreplication. Moreover, in order to amplify the number of gene copies inthe host cell system, the expression vector car. containphosphotransferase APH(3′)II or I (neo) gene, thymidine kinase (TX)gone, Escherichia coli xanthine-guanine phosphoribosyl transferase(Ecogpt) gene and dihydrofolic acid reductase (DHFR) and so forth asselection markers.

[0108] In addition, the present invention also provides a single-chainFv composed by linking an H chain V region and an L chain V region of areshaped hum antibody to human medulloblastoma cells. The H chain Vregion and L chain V region in this scFv polypeptide are preferablylinked by a linker, and more preferably, a peptide linker.

[0109] The H chain V region in the single-chain Fv may be any of the Hchain V regions of reshaped human antibody previously described.

[0110] An H chain V region comprises 4 FRs, and 3 CDRs having the aminoacid sequences defined below: CDR1; Asp Thr Tyr Ile His CDR2: Arg IleAsp Pro Ala Asp Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly CDR3: AlaTyr TyR Val Asn GLn Asp TyR

[0111] or a portion thereof.

[0112] In addition, an L chain V region comprises 4 FRs, and 3 CDRshaving the amino acid sequences defined below: CDR1: Lys Ala Ser Gln AsnVal Gly Thr Asn Val Ala CDR2: Ser Ala Ser Tyr Arg Tyr Ser CDR3: Gln GlnTyr Asn Ser Tyr Pro Arg Ala,

[0113] or a portion thereof.

[0114] A specific example of a single-chain Fv is that comprising an Hchain V region consisting of the amino acid sequence from amino acid 1to 116 in the amino acid sequence described in SEQ ID NO:80, and an Lchain V region consisting of the amino acid sequence from amino acid 1to 106 in the amino acid sequence described in any of SEQ ID NOs:40, 43,46, 47, 50. 51, 54, 55, 58, 61, 62,

[0115] In addition, a preferable example of a single-chain Fv is thatcomprising an H chain V region consisting of the amino acid sequencefrom amino acid 1 to 116 in the amino acid sequence described in SEQ IDNO:80, and an L chain V region consisting of the amino acid sequencefrom amino acid 1 to 106 in the amino acid sequence described in SEQ IDNO:73.

[0116] These V regions are preferably linked by polypeptide linkers.Although examples of polypeptide linkers include any single-chain Fvcomprising 12 to 19 amino acids, a specific example of a peptidefragment that can be used is the peptide fragment composed of Gly GlyGly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO.90).

[0117] An example of an amino acid sequence of a single-chain Fv isshown in SEQ ID NO:89. A single-chain Fv that possesses this amino acidsequence is referred to as scfv-hM21 in the present invention, and isexplained in detail in Example 6.

[0118] A DNA coding for the single-chain Fv of the present invention isobtained by using as a template a DNA coding for an H chain or H chain Vregion of a reshaped human ONS-M21 antibody and a DNA coding for an Lchain or L chain V region of reshaped human ONS-M21 antibody, both, ofwhich were previously explained in detail, and then amplifying a DNAportion coding for a desired amino acid sequence in those sequences byPCR using a pair of primers that specify both ends. Example 6 provides adetailed description of a method for preparing a DNA coding forsingle-chain Fv comprising an H chain V region and an L chain V regionversion “p”. Since the amino acid sequences of L chain V region versions“a” to “o” along with methods for preparing DNA coding for them aredescribed in detail, by applying a method in which version “p” is usedfor those aversions, DNA can be produced that code for varioussingle-chain Fvs of the present invention.

[0119] In addition, once DNA coding for single-chain Fv has beenproduced, expression vectors comprising that DNA as well as hoststransformed with said expression vectors can be obtained in accordancewith routine methods. In addition, single-chain Fvs can be obtained byusing those hosts in accordance with conventional methods. Specificexamples of these are described in detail in Example 6.

[0120] As a result of comparing the antigen binding ability of scFv-hM21with that of humanized ONS-M21 antibody and Fab fragment using for theindicator the degree of inhibition of binding of mouse ONS-M21 antibodyto ONS-76 cells, scFv-hM21 was found to exhibit binding inhibition equalto that of Fab fragment. On the basis of the above, a single-chain Fvwas able to be successfully constructed that possesses the same degreeof affinity as the original antibody.

[0121] In general, since single-chain Fvs are considered to exhibitsuperior mobility into tissue and tumors in comparison with whole IgG,this successfully constructed scFv-hM21 is expected to be used in thefuture in imaging by RI labeling as well as a therapeutic drug bycoupling with toxins or RI.

EXAMPLES

[0122] Next, although the following provides a specific explanation ofthe present invention through its Examples, the scope of the presentinvention is not limited by these Examples.

Example 1 Cloning of DNA Coding for the V Region of Mouse MonoclonalAntibody to Human Medulloblastoma Cell

[0123] DNA coding for a variable region of mouse monoclonal antibodyONS-M21 to human medulloblastoma cells was cloned in the followingmanner.

[0124] 1. Preparation of Messenger RNA (mRNA)

[0125] mRNA from hybridoma ONS-M21 was prepared using the Past TrackmRNA Isolation Kit Version 3.2 (Invitrogen).

[0126] 2. Synthesis of Double-Stranded cDNA

[0127] Double-stranded cDNA was synthesized using The Copy Kit(Invitrogen) from approximately 4 μg of mRNA.

[0128] 3. Amplification of Gene Coding for Antibody Variable Region byPCR

[0129] PCR was performed using Thermal Cycler (Perkin Elmer Cetus).

[0130] (1) Amplification of Gene Coding for Mouse L Chin V Region

[0131] MKV Mouse Kappa Variable) primers (Jones, S. T. et al.,Bio/Technology, 9, 88-89 (1991)) shown in SEQ ID NOs:1 to 11, whichhybridize with mouse kappa type L chain leader sequence, were used forthe primers used in PCR.

[0132] 100 μl of PCR solution contained 10 mM Triz-HCl (pH 8.3), 50 mMKCl, 0.1 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl₂, 5 units of DNApolymerase Ampli Taq (Perkin Elmer Cetus), 0.1 μM of MKV primer shown inSEQ ID NOs:1 to 11, 0.4 μM of MKC primer shown in SEQ ID NO: 12 and μgof double-stranded cDNA, and each of MCK primers 1 to 12 was separatelyamplified. After covering with 50 μl of mineral oil, the reactionmixture was heated at an initial temperature of 94° C. for 3 minutes andthen in the order of 94° C. for 1 minute, 50° C. for 1 minute and 72° C.for 1 minute. After repeating this temperature cycle 30 times, thereaction mixture was further incubated for 10 minutes at 72° C.

[0133] (2) Amplification of cDNA Coding for Mouse H Chain V Region

[0134] MHV (Mouse Heavy Variable) primers 1 to 12 shown in SEQ ID NOs;13to 24 and MHC-GI (Mouse Heavy Constant) primer (Jones, S. T. et al.,Bio/Technology, 9, 88-89 (1991)) shown in SEQ ID NO:25 were used for theprimers for PCR.

[0135] Amplification of cDNA was performed according to the same methodas that described for amplification of L chain V region gene in theabove-mentioned section 3(1) with the exception of performingamplification using a mixture of 0.25 μM car each MHV primer and 2.5 μMof MHC-GI primer.

[0136] 4. Purification and Cleavage of PCR Products

[0137] DNA fragments amplified by PCR as described above were purifiedwith low melting temperature agarose (Sigma) and digested for 3 hours at37° C. using 5 units of restriction enzyme XmaI (New England Biolabs) in10 m Tris-HCl (pH 7.9) containing 10 mM MgCl₂ and 1 mM dithreitol.

[0138] Next, after digesting for 2 hours at 37° C. with 40 units ofrestriction enzyme SalI (Takara Shuzo), the resulting DNA fragments wereseparated by agarose gal electrophoresis using 1.5% low meltingtemperature agarose (Sigma).

[0139] A piece of agarose containing a DNA fragment of approximately 450bp in length was cut out, melted for 5 minutes at 65° C. followed by theaddition of an equal volume of 20 mM Tris-HCl (pH 7.5) containing 2 mMEDTA and 200 mM NaCl. This mixture was extracted with phenol andchloroform, the DNA fragments were recovered by ethanol precipitationand then dissolved in 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA.

[0140] Thus, a DNA fragment comprising a gene coding for a mouse kappatype T chain variable region, amid a DNA fragment comprising a genecoding for a mouse H chain variable region were obtained. Theabove-mentioned DNA fragments have a SalI-cohesive end on their5′-terminal. and an XmaI-cohesive end on their 3′-terminal.

[0141] 5. Linkage and Transformation

[0142] Approximately 0.3 μg of SalI-XmaI DNA fragments comprising a genecoding for a mouse kappa type L chain V region prepared in the abovemanner were linked with approximately 0.1 μg of pUC19 vector prepared bydigesting with SalI and XmaI, by reacting for 4 hours at 16° C. in areaction mixture containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 10 mMdithiothreitol, 1 mM ATP, 50 m/ml of polyethylene glycol (8000) and 1unit of T4 DNA ligase (Gibco BRL).

[0143] Next, 10 μl of the above-mentioned linking mixture was added to50 μl of E. coli DH5α a competent cells after which the cells wereallowed to stand undisturbed for 30 minutes on ice, 1 minute at 42° C.and again for 1 minute on ice. Next, 400 μl of 2×Y medium (MolecularCloning: A Laboratory Manual, Sambrooks et al., Cold Spring HarborLaboratory Press, 1989) was added, and after incubating for 1 hour at37° C., 2×VT agar medium (Molecular Cloning: A Laboratory Manual,Sambrooks et al., Cold Spring Harbor Laboratory Press, 1989) wasinoculated with the E. coli and incubated overnight at 37° C. to obtainE. coli transformants.

[0144] These transformants were cultured overnight at 37° C. in 10 ml of2×YT medium containing 50 μg/ml of ampicillin, and plasmid DNA wasprepared from the culture according to the alkaline method (MolecularCloning: A Laboratory Manual, Sambrooks, et al., Cold Spring HarborLaboratory Press, 1989).

[0145] A plasmid containing a gene coding for a mouse kappa type L chainV region derived from hybridoma ONS-M21 obtained in this manner wasnamed pUC-M21-V_(L).

[0146] A plasmid containing a gene coding for a mouse H chain V regionderived from hybridoma ONS-M21 was made from SalI-XmaI DNA fragments inaccordance with the same method as that described above, and thatplasmid was named pUC-MS21-V_(H).

Example 2 Determination of DNA Nucleotide Sequence

[0147] A nucleotide sequence of a cDNA coding region in theabove-mentioned plasmid was determined in accordance with the protocolspecified by the manufacturer using an automated DNA sequencer (AppliedBiosystems Inc.) and the Taq Dye Deoxy Terminator Cycle Sequencing Kit(Applied Biosystem Inc.).

[0148] A nucleotide sequence of a gene coding for an L chain V region ofmouse ONS-N21 antibody contained in plasmid pUC-M21-V_(L) is shown inSEQ ID NO:26. In addition, a nucleotide sequence of a gene coding forthe H chain V region of mouse ONS-12 antibody contained in plasmidpUC-M21-V_(H) is shown in SEQ ID NO:27.

Example 3 Determination of CDR

[0149] The overall structures of the V regions of the L and H chainsmutually resemble each other, and 4 framework regions are linked by 3hypervariable regions, namely complementarity determining regions(CDRs). Although the amino acid sequence of the framework is relativelywell preserved, the variability of the amino acid sequence of the CDRsis extremely high (Kabat, E. A. et al., “Sequences of Proteins ofImmunological Interest”, US Dept. of Health and Human Services, 1983).

[0150] On the basis of these facts, the CDRs were determined as shown inTable 4 as a result of investigating homology by applying the amino acidsequence of the variable region of mouse monoclonal antibody to humanmedulloblastoma cells to the database of antibody amino acid sequencesprepared by Kabat, et al. TABLE 4 Plasmid SEQ ID NO CDR(1) CDR(2) CDR(3)pUC-M21-V_(L) 26 24-34 50-56 89-97 pUC-M21-V_(H) 27 31-35 50-66  99-106

Example 4 Confirmation of Expression of Cloned cDNA (Preparation ofChimeric ONS-M21 Antibody) Preparation of Expression Vector

[0151] In order to make vectors that express chimeric ONS-M21 antibody,cDNA clone pUC-M21-V_(L) and pUC-M21-V_(H) that respectively code forthe V regions of mouse ONS-M21κ L chain and H chain were modified byPCR. They were then introduced into REF expression vectors (see, thepreviously mentioned WO92-19759) (see, FIG. 1).

[0152] Backward primer ONS-L722S (SEQ D NO:28) for the L chair. V regionand backward primer ONS-H3.2S (SEQ ID NO:29) for the H chain V regionwere designed to hybridize with DNA coding for the start portion of theleader sequences of each V region and to have the Kozak consensussequence (Kozak, M. et al., J. Mol. Biol., 947-950, 1987) and an HindIIIrestriction site. Forward primer ONS-L722A (SEQ ID NO; 30) for the Lchain V region and forward primer ONS-H3.2A (SEQ ID NO:31) for the Hchain V region were designed to hybridize with DNA coding for the endportion of the J region and to have a splice donor sequence and BamHIrestriction site.

[0153] 100 μl of PCR reaction mixture containing 10 m Tris-HCl (pH 8.3),50 mM KCl, 100 μM dNTPs, 1.5 mM MgCl₂, 100 pmoles of each primer, 100 ngof template DNA (pUC-M²l-V_(L) or pUC-M²¹-V_(H)) and 5 units of AmpliTaq were covered with 50 μl of mineral oil. Following initialdenaturation at 94° C., an incubation cycle consisting of 1 minute at94° C., 1 minute at 55° C. and 1 minute at 72° C. was repeated 30 timesfollowed by final incubation for 10 minutes at 72° C.

[0154] The PCR products were purified using 5% low melting temperatureagarose gel, digested with HindIII and BamHI, and the L chain V regionwas cloned into HEF expression vector HEF-V_(L)-gκ, while the H chain Vregion was cloned into HEF expression vector HEF-V_(H)-gγ. Afterdetermining the DNA sequences, plasmids containing DNA fragments havingthe correct DNA sequence were respectively named HEF-M21L-gκ andHEF-M21H-gγ1.

Transfection into COS Cells

[0155] In order to observe the transient expression of chimeric ONS-M21antibody, the above-mentioned expression vectors were tested in COScells. HEF-M21L-gκ and HEF-M21H-gγ1 were co-transfected into COS cellsby electroporation using a Gene Pulser apparatus (BioRad). Each DNA (10μg) was added to an 0.8 ml aliquot of 1×10⁷ cells/ml in PBS followed bythe application of pulses at 1,900 V and capacitance of 25 μF.

[0156] After allowing a recovery period of 10 minutes at roomtemperature, the electroporated cells were added to DMEM culture mediumcontaining 10% γ-globulin-free Fetal calf serum (Gibco). Afterincubating for 72 hours, the culture supernatant was collected, celldebris was removed by centrifugation, and the supernatant was applied toa Protein A agarose column equilibrated with 5 volumes of binding buffer(Affi-Gel Protein A MAPSII Kit, BioRad). After washing the column with15 volumes of binding buffer, it was eluted with 5 volumes of elutionbuffer. The eluate was concentrated and the buffer was changed to PBSusing a microconcentrator (Centricon 100, Amicon).

Cell-ELISA

[0157] A Cell-ELISA plate for measuring antigen binding was prepared inthe following manner. Human medulloblastoma cell line ONS-76 (Tamura, etal., Cancer Res., 49, 5380-5384 (1989)) prepared to 1×10⁶ cells/ml withRPMI buffer containing 10% fetal calf serum was added to a 96-wellplate. After culturing overnight, the cells ware fixed with 0.1%glutaraldehyde (Nagai Chemical and Pharmaceuticals). After blocking,chimeric ONS-?21 antibody was serially diluted and added to each well.After incubating at room temperature and washing, alkalinephosphatase-bound goat anti-human IgG antibody (Sigma) was added. Afteradditional incubation and washing, substrate solution was added followedby measurement of absorbance at 405 nm.

[0158] As a result, it was suggested that chimeric antibody ONS-M21 hasthe correct stricture of the V region of mouse monoclonal antibodyONS-M21 since it specifically bound with medulloblastoma cell lineONS-76 (see, FIG. 2).

Example 5 Preparation of Reshaped Human ONS-N21 Antibody Preparation ofReshaped Human ONS-M21 Antibody Chain V Region

[0159] An L chain of a reshaped human ONS-M21 antibody was prepared byCDR-grafting using PCR. This technique is schematically illustrated inFIG. 3. In order to prepare a reshaped human antibody ONS-121 having FRsderived from human antibody REI (version “a”), 8 PCR primers were used.External primers A (SEQ ID NO:32) and H (SEQ ID NO:33) were designed soas to hybridize with the DNA sequence of HEF expression vectorHEF-V_(L)-gκ.

[0160] CDR-grafting primers B (SEQ ID NO:34), C (SEQ ID NO:35) and D(SEQ ID NO:36) have sense DNA sequences, while CDR-grafting primers E(SEQ ID NO:37)F (SEQ ID NO:39) and G (SEQ ID NO:39) have anti-sense DNAsequences, and have complementary DNA sequences (23-35 bp) to the DNAsequences of the 5′-terminals of primers B, C and D, respectively.

[0161] In the first stage of PCR, the four reactions between A-E, B-F,C-G and D-H were performed, and each PCR product was purified. The fourPCR products from the first stage of PCR were assembled according totheir own complementarity (see, WO92-19759). Next, external primers Aand H were added to amplify the entire DNA coding for an L chain Vregion of a reshaped human ONS-M21 antibody (second stage of PCR). Inthe above-mentioned PCR, plasmid HEF-RV_(L)-SK2a coding for version “a”of the L chain V region of reshaped human SK2 antibody based on the FRsfrom human antibody REI (see, Japanese Unexamined Patent Publication No.5-129787) was used as a template.

[0162] In the first stage of PCR, 100 μl of PCR mixture containing 50 mMKCl, 100 μM dNTPs, 1.5 mM MgCl₂, 100 ng of template DNA, 100 pmoles ofeach primer and 5 units of Ampli Taq were used. Each PCR tube wascovered with 50 μl of mineral oil. After initially denaturing at 94° C.,a reaction cycle was performed consisting of 1 minute at 94° C., 1minute at S5° C. and 1 minute at 72° C. followed finally by incubationfor 10 minutes at 72° C.

[0163] PCR products A-E (218 bp), B-F (101 bp), C-G (131 bp) and D-H(147 bp) were purified using 1.5% low melting temperature agarose gel,and assembled in the second stags of PCR. In the second stage of PCR, 98of PCR mixtures containing 1 μg of each first stage PCR product and 5units of Ampli Taq were incubated in a cycle consisting of 2 minutes at94° C., 2 minutes at 55° C. and 2 minutes at 72° C. after which 100pmoles of each external primers (A and H) were added. The PCR tube wascovered with 50 μl of mineral oil and 30 cycles of PCR were performedunder the same conditions as described above.

[0164] The 516 bp DNA fragment produced from the second stage of PCR waspurified with 1.5% low melting temperature agarose gel, digested withBamHI and HindIII, and the resulting DNA fragment was cloned into HEFexpression vector HEF-V_(L)-gκ. After determining the DNA sequence, theplasmid containing the DNA fragment coding for the correct amino acidsequence of the L chain V region of a reshaped human ONS-M21 antibodywas named HEF-RVL-M21a-gκ. The amino acid sequence and nucleotidesequence of the L chain V region contained in this plasmidHEF-RVL-M21a-gκ are shown in SEQ ID NO:40.

[0165] Version “b” of the L chain V region of reshaped human ONS-Mlantibody was prepared by PCR mutagenesis. Mutagenic primers FTY-1 (SEQID NO:41) and FTY-2 (SEQ ID NO:42) were designed so that thephenylalanine at position 71 is substituted to tyrosine.

[0166] After amplification using the above-mentioned primers and plasmidHEF-RVL-M21-a-gκ as a template, the final product was purified, digestedwith BamHI and HindIII, and the resulting DNA fragment was cloned intoHEF expression vector HEF-VL-gκ to obtain plasmid HEF-RVL-21b-gκ. Theamino acid sequence and nucleotide sequence of the L chain V regioncontained in this plasmid HEF-RVL-M21b-gκ are shown in SEQ ID NO:43.

[0167] Each of versions “c”, “d”, “e”, “f”, “g”, “h”, “i”, “j”, “k”,“l”, “m”, “n”, “o” and “p” of the L chain V region of reshaped humanONS-M21 antibody was produced in the manner described below.

[0168] For version “c”, amplification was performed by PCR using primersM21M2S (SEQ ID NO:44) and M21M:)A (SEQ ID NO:45), which were designed sothat tyrosine at position 87 is substituted to phenylalanine, asmutagenic primers, and using plasmid HEF-RVL-M21a-gκ as a template DNAto obtain plasmid HEF-RVL-M21c-gκ. The amino acid sequence andnucleotide sequence of the L chain V region contained in this plasmidHEF-RVL-M21c-gκ are shown in SEQ ID NO:46.

[0169] For version “d”, amplification was performed using primers FTY-1and FTY-2 as well as M21M2S and M21M2A as mutagenic primers, and usingplasmid HEF-RVL-M21a-gκ as a template DNA to obtain plasmidHEF-RVL-M21d-gκ. The amino acid sequence and nucleotide sequence of theL chain V region contained in this plasmid HEF-RVL-M21d-gκ are shown inSEQ ID NO:47.

[0170] For version “e”, amplification was performed using primers M21M3S(SEQ ID NO:48) and M21M3A (SEQ ID NO:45), which were designed so thatthreonine at position 20 is substituted to serine and isoleucine atposition 21 is substituted to valine, as mutagenic primers, and usingplasmid HEF-RVL-M21a-gκ as a template DNA to obtain plasmidHEF-RVL-M21e-gκ. The amino acid sequence and nucleotide sequence of theL chain V region contained in his plasmid HEF-RVL-M21e-gκ are shown inSEQ ID NO:50.

[0171] For version “f”, plasmid HEF-RV_(L)-M21c-gκ was digested withBamHI and HinfI, and plasmid HEF-RV-M21e-gκ was digested with HindIIIand HinfI to obtain 152 bp and 250 bp DNA fragments respectively. Afterisolating and purifying these DNA fragments using 15% low meltingtemperature agarose gel, the fragments were linked and inserted into HEFexpression vector HEF-RVL-gκ to obtain plasmid HEF-RVL-M21f-gκ. Theamino acid sequence and nucleotide sequence of the L chain V regioncontained in this plasmid HEF-RVL-M21f-gκ are shown in SEQ ID NO:51.

[0172] For version “g”, amplification was performed using primers M21M4S(SEQ ID NO:52) and M21M4A (SEQ ID NO:53), which were designed so thatphenylalanine at position 73 is substituted to leucine, as mutagenicprimers, and using plasmid HEF-RVL-M21f-gκ as a template DNA to obtainplasmid REF-RVL-M21g-gκ. he amino acid sequence and nucleotide sequenceof the L chain V region contained in this plasmid HEF-RVL-M21gκ areshown in SEQ ID NO:54.

[0173] For version “h”, amplification was performed using primers M21M4Sand M21M4A as mutagenic primers, and using plasmid HEF-RVL-M21a-gκ as atemplate DNA to obtain plasmid HEF-RV-M21h-gκ. The amino acid sequenceand nucleotide sequence of the L chain V region contained in thisplasmid HEF-RVL-M21h-gκ are shown in SEQ ID NO:55.

[0174] For version “i”, amplification was performed using primers M21M5S(SEQ ID NO:56) and M21M5A (SEQ ID NO:57), which were designed so thatlysin. at position 42 is substituted to glutamine, analine at position43 is substituted to serine and leucine at position 46 is substituted toproline, as mutagenic primers, and using plasmid HEF-RVL-M21g-gκ an atemplate DNA to obtain plasmid HEF-RVL-M21i-gκ. The amino acid sequenceand nucleotide sequence of the L chain V region contained in thisplasmid REF-RVL-M21i-gκ are shown in SEQ ID NO:58, For version “j”,amplification was performed using primers M21M5S, M21M5A as well asM21M6S (SEQ ID NO:59) and M21M6A (SEQ ID NO:60), which were designed sothat threonine at position 85 is substituted to aspartate, as mutagenicprimers, and using plasmid HEF-RVL-M21i-gκ as a template DNA to obtainplasmid HEF-RVL-M21j-gκ. The amino acid sequence and nucleotide sequenceof the L chain V region contained in this plasmid HEF-RVL-M21j-gκ areshown in SEQ ID NO:61.

[0175] For version “k”, amplification was performed using primers M21M6Sand M21M6A as mutagenic primers, and using plasmid HEF-RVL-M21g-gκ as atemplate DNA to obtain plasmid HEF-RVL-M21k-gκ. The amino acid sequenceand nucleotide sequence of the L chain V region contained in thisplasmid HEF-RVL-M21k-gκ are shown in SEQ ID NO:62.

[0176] For version “l” plasmid HEF-RVL-M21a-gκ was digested with BamHIand SfaNI, and plasmid HEF-RVL-M21i-gκ was digested with HindIII andSfaNI to obtain 227 bp and 169 bp DNA fragments respectively. Afterisolating and purifying these DNA fragments, the resulting DNA fragmentswere linked and inserted into HEF expression vector HEF-RVL-gκ to obtainplasmid HEF-RVL-M21l-gκ. The amino acid sequence and nucleotide sequenceof the L chain V region contained in this plasmid HEF-RVL-M21l-gκ areshown in SEQ ID NO:63.

[0177] For version “m”, amplification was performed using primersM21M5S, M21M5A as well as M21M7S (SEQ :D NO:64) and M21M7A (SEQ IDNO:65), which were designed so that proline at position 8 is substitutedto glutamate, serine at position 9 is substituted to lysine and serineat position 10 is substituted to phenylalanine, as mutagenic primers,and using plasmid HEF-RVL-M21a-gκ as a template DNA to obtain plasmidHEF-RVL-M21m-gκ. The amino acid sequence and nucleotide sequence of theL chain V region contained in this plasmid HEF-RVL-M21m-gκ are shown inSEQ ID NO:66.

[0178] For version “n”, amplification was performed using primers M21M8S(SEQ ID NO:67) and M21M8A (SEQ ID NO:68), which were designed so thatlysine at position 42 is substituted to glutamate and alanine atposition 43 is substituted to serine, as mutagenic primers, and usingplasmid HEF-RVL-M21a-gκκ as a template DNA to obtain plasmidHEF-RVL-M21n-gκ. The amino acid sequence and nucleotide sequence of theL chain V region contained in this plasmid HEF-RVL-M21n-gκ are shown inSEQ ID NO:69.

[0179] For version “o”, plasmid HEF-RVL-M21b-gκ was digested with BamHIand BsrI, and plasmid HEF-RVL-M21a-gκ was digested with HindIII and BsrIto obtain 251 bp and 142 bp DNA fragments respectively. After isolatingand purifying these DNA fragments, the resulting DNA fragments werelinked and inserted into HEF expression vector HEF-RVL-gκ to obtainplasmid HEF-RVL-M21o-gκ. The amino acid sequence and nucleotide sequenceof the L chain V region contained in this plasmid HEF-RVL-M21o-gκ areshown in SEQ ID NO:70.

[0180] For version “p”, amplification was performed using primers M21M9S(SEQ ID NO:71) and M21M9A (SEQ ID NO:72), which were designed so thatleucine at position 46 is substituted to proline, as mutagenic primers,and using plasmid HEF-RVL-M21a-gκ as a template DNA to obtain plasmidHEF-RVL-M21p-gκ. The amino acid sequence and nucleotide sequence of theL chain V region contained in this plasmid HEF-RVL-M21p-gκ are shown inSEQ ID NO:73.

Preparation of Reshaped Human ONS-M21Antibody H Chain V Region

[0181] DNA coding for the K chain V region of reshaped human ONS-M21antibody was designed in the following manner. DNA sequences coding forFR1-3 of human antibody Eu and FR4 of human antibody ND were designedbased on “codon usage” of the V region (Kabat, E. A. et al., US Dept. ofHealth and Human Services, US Government Printing Offices, 1991). Byconnecting with a DNA sequence coding for the CDRs of the H chain Vregion of mouse ONS-M21 antibody, an entire length of DNA was designedcoding for the H chain V region of reshaped human ONS-M21 antibody.

[0182] Next, a HindIII recognition site/KOZAK consensus sequence andBamHI recognition site/splice donor sequence were added to this5′-terminal and 3′-terminal, respectively, of this DNA sequence toenable inserted into an HEF expression vector.

[0183] The DNA sequence designed in this manner was then divided intofour oligonucleotides, and the secondary structures of oligonucleotideshaving the potential to inhibit assembly of these oligonucleotides wereanalyzed by computer.

[0184] The four oligonucleotide sequences are shown in SEQ ID NO:74 to77. These oligonucleotides have lengths of 111 to 137 bases, and possessoverlapping regions of 23 to 26 bp. Among the oligonucleotides, the RVH2(SEQ ID NO:74) and RVH4 (SEQ ID NO:75) have sense DNA sequences, whilethe other RVHI (SEQ ID NO:76) and RVH3 (SEQ ID NO:77) have anti-senseDNA sequences. The assembly method of these four oligonucleotides by PCRis shown in the illustration (see, FIG. 4).

[0185] After initially denaturing 98 μl of PCR mixture containing 100 ngof each of the four types of oligonucleotides and 5 units of Ampli Taqfor 2 minutes at 94° C., the mixture was incubated for 2 cyclesconsisting of 2 minutes at 94° C., 2 minutes at 55° C. and 2 minutes at72° C. After adding 100 pmoles each of RHP1 (SEQ ID NO:78) and RHP2 (SEQID NO:79) as external primers, the PCR tube was covered with 50 μl ofmineral oil, and after initially denaturing for 1 minute at 94° C., 38cycles of incubation consisting of 1 minute at 94° C., 1 minute at 55°C. and 1 minute at 72° C. were performed followed by final incubationfor 10 minutes at 72° C.

[0186] The 438 bp DNA fragment was purified using 1.5% low meltingtemperature agarose gel, digested with HindIII and BamHI, and cloned inHEF expression vector HEF-V_(H)-gγ1. After determining the DNA sequence,the plasmid containing the DNA fragment coding for the correct aminoacid sequence of the H chain V region was named HEF-RVH-M21-gγ1. Theamino acid sequence and nucleotide sequence of the H chain V regioncontained in this plasmid REF-RVH-M21-gγ1 are shown in SEQ ID NO:80.

[0187] In order to evaluate each chain of reshaped human ONS-M21antibody, COS cells were co-transfected as previously described withexpression vector HEF-RVH-M21-gγ1 for the H chain of reshaped humanONS-M21 antibody, and expression vector HEF-M21L-gγ1 for the L chain ofchimeric ONS-M21 antibody. After collecting the antibody products aspreviously described, antigen binding was measured as previouslydescribed.

[0188] Those results are shown in FIG. 5. As shown in FIG. 5, there wasconfirmed to be no difference in antigen binding between chimericantibody (ChM21) used as positive control and antibody comprisingreshaped H chain and chimeric L chain (ChL/RVH) .

[0189] Next, in order to evaluate combinations of each of versions “a”through “p” of reshaped humanized ONS-M21 antibody L chain and reshapedhumanized ONS-M21 antibody H chain, one of each of the expressionplasmids from HEF-RVL-M21a-gκ to HEF-RVH-M21p-gκ of each version of theL chain and H chain expression plasmid HEF-RVH were co-transfected intoCOS cells and antigen binding was measured for the resulting antibodiesusing the method as described in Cell ELISA of the above-mentionedEmbodiment 4. As a result, antigen binding activity was not observed inantibodies having L chain versions “a”, through “h” (see, FIG. 6).

[0190] On the other hand, antibodies having each of the L chain versions“i”, “j”, “l”, “m”, “o” and “p” demonstrated good antigen bindingcomparable to that of the positive control chimeric ONS-M21 antibody(ChM21), and this combination was suggested to create a functionalantigen binding site in human antibody. Furthermore, antigen bindingactivity was not observed in antibodies having L chain versions Ski and”n” (refer to FIG. 7).

[0191] On the basis of these findings, antibody having proline atposition 46 of the L chain FR2 was suggested to recreate a functionalantigen binding site that exhibits good antigen binding.

Example 6 Preparation of Reshaped Human ONS-M21 Antibody Single-Chain Fv(scFV) Construction of Linker Region

[0192] DNA coding for a linker region consisting of Gly Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO:90) was designed inthe following runner. A 15 bp DNA sequence coding for 5 amino acidresidues of the C-terminal of FR4 of the H chain V region was added tothe 5′-terminal of a DNA sequence coding for a linker region (Huston, J.S. et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988)), while a 15bp DNA sequence coding for 5 amino acid residues of the N-terminal ofthe FRI region of the L chain V region was added to the 3′-terminal.Moreover, HindIII and EcoRI recognition sites were added to the5′-terminal and 3′-terminal, respectively, to enable insertion intopUC19 vector.

[0193] Two oligonucleotides scFv-S and scFV-A in the sense andanti-sense directions were synthesized based on the DA sequencesdesigned in this manner.

[0194] The two oligonucleotide sequences are shown in. SEQ ID NOs:81 and82, respectively. These oligonucleotides have a length of 84 bases, andhave an overlapping region of 81 bp.

[0195] 100 pmoles each of the two oligonucleotides were placed in 20 μlof a reaction mixture containing 50 mM Tris-HCl (pH 7.6), 10 m MgCl₂, 10nM dithiothreitol, 1 mM ATP and 50 mg/ml polyethylene glycol (8000).Annealing was performed by incubating for 5 minutes at 96° C., loweringthe temperature to 65° C. over the course of 20 minutes, incubating for10 minutes at 65° C., lowering the temperature to 37° C. over the courseof 20 minutes, incubating for 10 minutes at 37° C., lowering thetemperature to 70° C. over the course of 20 minutes and finallyincubating at 7° C. overnight.

[0196] The DNA annealed in the above-mentioned manner was inserted intopUC19 vector cleaved with HindIII and EcoRI. After determining the DNAsequence, the plasmid containing the DNA fragment that has the correctDNA sequence was named pUC-scFv-5.

Preparation of Reshaped Human ONS-M21 Antibody7 Single-Chain Fv

[0197] The single-chain Fv of reshaped human ONS-M21 antibody wasproduced in the following manner. Reshaped human ONS-M21 antibody Hchain V region, linker region and reshaped human ONS-M21 antibody Lchain V region were respectively amplified and assembled using PCR toconstruct reshaped human ONS-M21 antibody single-chain Fv. Thisprocedure is schematically illustrated in FIG. 8. Six PCR primers (A toF) were used to construct the single-chain Fv of reshaped human ONS-M21antibody. Primers A, C and E have sense sequences, while primers B, Dand F have anti-sense sequences.

[0198] Backward primer SCP1 (primer A, SEQ ID NO:83) for the H chain Vregion was designed to hybridize with DNA coding for the N-terminal ofthe H chain V region and to have an NcoI recognition site. Forwardprimer SCP2 (primer B, SEQ ID NO:84) for the H chain V region wasdesigned to hybridize with DNA coding for the C-terminal of the H chainV region and to overlap with the linker. Backward primer SCP3 (primer C,SEQ ID NO:85) for the linker region was designed to hybridize with DNAcoding for the N-terminal of the linker and to overlap with DNA codingfor the C-terminal of the H chain V region.

[0199] Forward primer SCP4 (primer D, SEQ ID NO:86) for the linkerregion was designed to hybridize with DNA coding for the C-terminal ofthe linker and to overlap with DNA coding for the N-terminal of the Lchain V region. Backward primer SCP4 (primer E, SEQ ID NO:87) for the Lchain V region was designed to hybridize with DNA coding for theC-terminal of the linker and to overlap with DNA coding for theN-terminal of the L chain V region. Forward primer WS4-2 (primer F. SEQID NO:88) for the L chain V region was designed to hybridize with DNAcoding for the C-terminal of the L chain V region and to have a sequencecoding for the FLAG peptide (Hopp, T. P. et al., Bio/Technology, 6,1204-1210, 1988), two transcription termination codons and an EcoRIrecognition site.

[0200] In the first step of PCR, three reactions were conducted betweenA-B, C-D and E-F and each PCR product was. purified. The three PCRproducts from the first step of PCR were assembled according to theirown complementarity. Next, primers A and F were added to amplify theentire length of DNA coding for the single-chain Pv of reshaped humanONS-M21 antibody (second step of PCR). Furthermore, in the first step ofPCR, plasmid HEF-RV-M21-gγ1 coding for the H chain V region of reshapedhuman ONS-M21 antibody (refer to Embodiment 5). plasmid pUC-scFv-5coding for the linker region, and plasmid HEF-RV_(L)-M21p-gκ (refer toEmbodiment 5) coding for version “p” of the L chain V region of reshapedhuman ONS-M21 antibody were respectively used as templates.

[0201] In the first step of PCR, a PCR mixture was used containing 10 mmTris-HCl (pH 8.3), 50 mM KCl, 100 μM dNTPs, 1.5 mM MgCl₂, 100 ng of eachtemplate DNA, 100 pmoles of each primer and 5 units of DNA polymeraseAmpli Taq (Perkin Elmer Cetus). After covering each PCR tube with 50 μlof mineral oil, the tube was heated in the order of 1 minute at 94° C.,1 minute at 55° C. and 1 minute at 72° C. After repeating thistemperature cycle 30 times, the reaction mixture was additionallyincubated for 10 minutes at 72° C.

[0202] PCR products A-B (382 bp), C-D (92 bp) and E-F (363 bp) werepurified using 1.5% low melting temperature agarose gel and assembled inthe second step of PCR. In the second step of PCR, 98 μI of PCR reactionmixture, containing 1 μg of each of the first stage PCR products astemplates and 5 units of Ampli Taq, were incubated for 2 cyclesconsisting of 2 minutes at 94° C., 2 minutes at 55° C. and 2 minutes at72° C. followed by addition of 100 pmoles each of primers A and F. Aftercovering the PCR tube with 50 μl of mineral oil, 30 cycles of PCR wereperformed consisting of 1 minute at 94° C., 1 minute at 55° C. and 2minutes at 72° C.

[0203] The 767 bp DNA fragment produced in the second step of PCR waspurified with 1.5!blow melting temperature agarose gel and digested withNcoI and EcoRI, after which the resulting DNA fragments were cloned inexpression vector pSCFVT7. Furthermore. this expression vector pSCFVT7contains a pelB signal sequence suited to E. coli periplasm secretionexpression systems (Lei, S. P. et al., J. Bacteriology, 169, 4379-4383,1987). After determining the DNA sequence, the plasmid containing theDNA fragment coding for the correct amino acid sequence of thesingle-chain Fv of reshaped human ONS-M21 antibody was namedpSCFVT7-hH21 (see FIG. 9). The amino acid sequence and nucleotidesequence of the single-chain Tv of reshaped human ONS-M21 antibodycontained in this plasmid SCFVT7-hM21 are shown in SEQ ID NO:89.

Transformation of E. coli Strain BL21 (DE3)

[0204] 10 ng of the above-mentioned plasmid pSCFVT7-hM21 was added to 50μl of E. coli BL21(DE3) competent cells, after which the cells wereallowed to stand for 30 minutes on ice, for 90 seconds at 42° C. andagain for 1 minute on ice. Next, 400 μl of 2×YT medium was added andafter incubating for 1 hour at 37° C., the E. coli was plated onto 2×YTagar medium and incubated overnight at 37° C. to obtain E. colitransformants.

Induction of Expression of Reshaped Human ONS-M21 Antibody Single-ChainFv Region

[0205] The transformed E. coli was cultured overnight at 37° C. in 30 mlof LB medium (Molecular Cloning: A Laboratory Manual, Sambrook, et al.,Cold Spring Harbor Laboratory Press, 1989) containing 1% glucose and 50μg/ml ampicillin. Next, the culture was innoculated into 100 timesvolume of LB medium containing 50 μg/ml ampicillin and cultured at 37°C. Isopropyl thio-β-D-galactoside (IPTG) was added to a finalconcentration of 0.5 mM when absorbance at 650 nm reached about 0.3 toinduce expression from T7 promoter.

[0206] After additionally culturing overnight at 37° C., the medium wascollected, cell debris was removed by centrifugation followed by theaddition of an equal volume of PBS. After equilibrating with 15 ml of0.1 M glycine-HCl (pH 3.0), the medium was applied to an anti-FLAGaffinity column neutralized with an equal volume of PBS (Anti-FLAGM2Affinity Gel, IBI). After washing the column with 3 volumes of PBS, thecolumn was eluted with 6 ml of 0.1 M glycine-HCl (pH 3.0). The eluatewas concentrated and the buffer was changed to PBS using amicroconcentrator (Centricon10, Amicon).

Cell-ELISA

[0207] The antigen binding activity of the single-chain Fv of reshapedhuman ONS-M21 antibody was measured using for an indicator an inhibitoryactivity of mouse monoclonal ONS-M21 antibody on antigen binding. Afterblocking the Cell-ELISA plate prepared as described above, seriallydiluted samples of the reshaped human ONS-M21 antibody single-chain Fv,reshaped human ONS-K21 antibody or of Fab fragment prepared from thereshaped human ONS-M21 antibody were added together with mousemonoclonal ONS-N:21 antibody at a concentration of 500 ng/ml to eachwell. After incubating at room temperature and washing,alkaline-phosphatase-bound goat anti-mouse IgG antibody (Zymed) wasadded After incubating and washing, substrate solution was addedfollowed by measurement of absorbance at 405 nm.

[0208] As a result, although inhibitory activity of the single-chain Fvof reshaped human ONS-M21 antibody (scFv-hM21) decreased to about{fraction (1/10)} in comparison with reshaped human ONS-M21 antibody(hM21), it showed almost same inhibitory activity as the Fab fragmentprepared from reshaped human ONS-M21 antibody {see, FIG. 10}.

[0209] On the basis of these findings, single-chain Fv of a reshapedhuman ONS-M21 antibody was suggested to exhibit roughly the same degreeof affinity as the original reshaped human ONS-M21 antibody.

[0210] Furthermore, E. coli having the above-mentioned plasmidHEF-RVL-M21p-gκ and E. coli containing plasmid HEF-RVH-M21-gγ arerespectively deposited as Escherichia coli DH5α (HEF-RVL-M21p-gκ) andEscherichia coli DH5α (HEF-RVH-M21-gγ1) at the National Institute ofBioscience and Human Technology Agency of Industrial Science andTechnology (1-3 Higashi, 1-chome Tsukuba-shi, Ibaraki), and weresubmitted for international deposit under the provisions of the BudapestAccord as FERM BP-4472 and FERM BP-4471 on Nov. 18, 1993.

Reference Example 1 Preparation of Hybridoma ONS-M21

[0211] Hybridoma that produces an anti-human medulloblastoma cellmonoclonal antibody was prepared by fusing spleen cells of a BALB/cmouse immunized with human medulloblastoma cells ONS-76 and mousemyeloma cells P3U1 in accordance with routine methods using polyethyleneglycol. Screening was performed using as an indicator a binding activitywith medulloblastoma cells ONS-76 so as to establish hybridoma ONS-M21(Moriuchi, S. et al., Br. J. Cancer, 68, 831-837 (1993)).

Reference Example 2 Typing of Mouse Monoclonal Antibody ONS-M21

[0212] Hybridoma ONS-M21 was transplanted into mouse abdominal cavityand the resulting ascites was applied to a Protein A agarose column toobtain purified mouse monoclonal antibody. In order to investigate thetypes of the L chain and H chain of the resulting mouse monoclonalantibody ONS-M21, typing was performed using a mouse monoclonal antibodyisotyping kit (Amersham international Plc.). As a result, ONS-M21antibody was clearly shown to have a κ K type L chain and γ1 type Hchain.

[0213] Reference to Microorganisms Deposited Based on Provision 13, Part2 of the Budapest Treaty;

[0214] Deposition institution: National Institute of Bioscience andHuman Technology, Agency of Industrial Science and Technology

[0215] Address: 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki DepositionNumbers and Dates:

[0216] 1. Escherichia coli DH5α (HEF-RVL-M21p-gκ) Deposition no.: FERMBP-4472 Deposition date: Nov. 18, 1993

[0217] 2. Escherichia coli DH5α (HEF-RVH-M21-Tγ1) Deposition no.: FERMBP-4471 Deposition date: Nov. 18, 1993

1 132 40 base pairs nucleic acid single linear 1 ACTAGTCGAC ATGAAGTTGCCTGTTAGGCT GTTGGTGCTG 40 39 base pairs nucleic acid single linear 2ACTAGTCGAC ATGGAGWCAG ACACACTCCT GYTATGGGT 39 40 base pairs nucleic acidsingle linear 3 ACTAGTCGAC ATGAGTGTGC TCACTCAGGT CCTGGSGTTG 40 43 basepairs nucleic acid single linear 4 ACTAGTCGAC ATGAGGRCCC CTGCTCAGWTTYTTGGMWTC TTG 43 40 base pairs nucleic acid single linear 5 ACTAGTCGACATGGATTTWC AGGTGCAGAT TWTCAGCTTC 40 37 base pairs nucleic acid singlelinear 6 ACTAGTCGAC ATGAGGTKCY YTGYTSAGYT YCTGRGG 37 41 base pairsnucleic acid single linear 7 ACTAGTCGAC ATGGGCWTCA AGATGGAGTC ACAKWYYCWGG 41 41 base pairs nucleic acid single linear 8 ACTAGTCGAC ATGTGGGGAYCTKTTTYCMM TTTTTCAATT G 41 35 base pairs nucleic acid single linear 9ACTAGTCGAC ATGGTRTCCW CASCTCAGTT CCTTG 35 37 base pairs nucleic acidsingle linear 10 ACTAGTCGAC ATGTATATAT GTTTGTTGTC TATTTCT 37 38 basepairs nucleic acid single linear 11 ACTAGTCGAC ATGGAAGCCC CAGCTCAGCTTCTCTTCC 38 27 base pairs nucleic acid single linear 12 GGATCCCGGGTGGATGGTGG GAAGATG 27 37 base pairs nucleic acid single linear 13ACTAGTCGAC ATGAAATGCA GCTGGGTCAT STTCTTC 37 36 base pairs nucleic acidsingle linear 14 ACTAGTCGAC ATGGGATGGA GCTRTATCAT SYTCTT 36 37 basepairs nucleic acid single linear 15 ACTAGTCGAC ATGAAGWTGT GGTTAAACTGGGTTTTT 37 35 base pairs nucleic acid single linear 16 ACTAGTCGACATGRACTTTG GGYTCAGCTT GRTTT 35 40 base pairs nucleic acid single linear17 ACTAGTCGAC ATGGACTCCA GGCTCAATTT AGTTTTCCTT 40 37 base pairs nucleicacid single linear 18 ACTAGTCGAC ATGGCTGTCY TRGSGCTRCT CTTCTGC 37 36base pairs nucleic acid single linear 19 ACTAGTCGAC ATGGRATGGAGCKGGRTCTT TMTCTT 36 33 base pairs nucleic acid single linear 20ACTAGTCGAC ATGAGAGTGC TGATTCTTTT GTG 33 40 base pairs nucleic acidsingle linear 21 ACTAGTCGAC ATGGMTTGGG TGTGGAMCTT GCTATTCCTG 40 37 basepairs nucleic acid single linear 22 ACTAGTCGAC ATGGGCAGAC TTACATTCTCATTCCTG 37 38 base pairs nucleic acid single linear 23 ACTAGTCGACATGGATTTTG GGCTGATTTT TTTTATTG 38 37 base pairs nucleic acid singlelinear 24 ACTAGTCGAC ATGATGGTGT TAAGTCTTCT GTACCTG 37 28 base pairsnucleic acid single linear 25 GGATCCCGGG CCAGTGGATA GACAGATG 28 382 basepairs nucleic acid double linear CDS 1..381 26 ATG GAG TCA CAT ATT CAGGTC TTT GTA TAC ATG TTG CTG TGG TTG TCT 48 Met Glu Ser His Ile Gln ValPhe Val Tyr Met Leu Leu Trp Leu Ser 1 5 10 15 GGT GTT GAT GGA GAC ATTGTG ATG ACC CAG TCT CAA AAA TTC ATG TCC 96 Gly Val Asp Gly Asp Ile ValMet Thr Gln Ser Gln Lys Phe Met Ser 20 25 30 ACA TCA GTA GGA GAC AGG GTCAGC GTC ACC TGC AAG GCC AGT CAG AAT 144 Thr Ser Val Gly Asp Arg Val SerVal Thr Cys Lys Ala Ser Gln Asn 35 40 45 GTG GGT ACT AAT GTA GCC TGG TATCAA CAG AAA CCA GGG CAA TCT CCT 192 Val Gly Thr Asn Val Ala Trp Tyr GlnGln Lys Pro Gly Gln Ser Pro 50 55 60 AAA CCA CTG ATT TAC TCG GCA TCC TATCGG TAC AGT GGA GTC CCT GAT 240 Lys Pro Leu Ile Tyr Ser Ala Ser Tyr ArgTyr Ser Gly Val Pro Asp 65 70 75 80 CGC TTC ACA GGC AGT GGA TCT GGG ACAGAT TTC ACT CTC ACC ATC ACC 288 Arg Phe Thr Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Thr 85 90 95 AAT GTG CAG TCT GAA GAC TTG GCA GAC TATTTC TGT CAG CAA TAT AAC 336 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr PheCys Gln Gln Tyr Asn 100 105 110 AGC TAT CCT CGG GCG TTC GGT GGA GGC ACCAAA CTG GAA ATC AAA 381 Ser Tyr Pro Arg Ala Phe Gly Gly Gly Thr Lys LeuGlu Ile Lys 115 120 125 C 382 127 amino acids amino acid linear protein27 Met Glu Ser His Ile Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser 1 510 15 Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser 2025 30 Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn 3540 45 Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 5055 60 Lys Pro Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp 6570 75 80 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr85 90 95 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn100 105 110 Ser Tyr Pro Arg Ala Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys115 120 125 409 base pairs nucleic acid single linear CDS 1..408mat_peptide 1..408 28 ATG AAA TGC AGC TGG GTC ATG TTC TTC CTG ATG GCAGTG GTT ACA GGG 48 Met Lys Cys Ser Trp Val Met Phe Phe Leu Met Ala ValVal Thr Gly 1 5 10 15 GTC AAT TCA GAG GTT CAG CTG CAG CAG TCT GGG GCAGAG CTT GTG AAG 96 Val Asn Ser Glu Val Gln Leu Gln Gln Ser Gly Ala GluLeu Val Lys 20 25 30 CCA GGG GCC TCA GTC AAG TTG TCC TGC ACA GCT TCT GGCTTC AAC ATT 144 Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly PheAsn Ile 35 40 45 AAA GAC ACC TAT ATA CAC TGG GCG AAG CAG AGG CCT GAA CAGGGC CTG 192 Lys Asp Thr Tyr Ile His Trp Ala Lys Gln Arg Pro Glu Gln GlyLeu 50 55 60 GAG TGG ATT GGA AGG ATT GAT CCT GCG GAT GGT AAT ACT AAA TATGAC 240 Glu Trp Ile Gly Arg Ile Asp Pro Ala Asp Gly Asn Thr Lys Tyr Asp65 70 75 80 CCG AAG TTC CAG GGC AAG GCC ACT ATA ACA GCA GAC ACA TCC TCCAAC 288 Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn85 90 95 ACA GCC TAC CTG CAG CTC AGC AGC CTG ACA TCT GAG GAC ACT GCC GTC336 Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100105 110 TAT TAC TGT GCT TCG GCC TAC TAT GTT AAC CAG GAC TAC TGG GGT CAA384 Tyr Tyr Cys Ala Ser Ala Tyr Tyr Val Asn Gln Asp Tyr Trp Gly Gln 115120 125 GGA ACC TCA GTC ACC GTC TCC TCA G 409 Gly Thr Ser Val Thr ValSer Ser 130 135 136 amino acids amino acid linear protein 29 Met Lys CysSer Trp Val Met Phe Phe Leu Met Ala Val Val Thr Gly 1 5 10 15 Val AsnSer Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys 20 25 30 Pro GlyAla Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile 35 40 45 Lys AspThr Tyr Ile His Trp Ala Lys Gln Arg Pro Glu Gln Gly Leu 50 55 60 Glu TrpIle Gly Arg Ile Asp Pro Ala Asp Gly Asn Thr Lys Tyr Asp 65 70 75 80 ProLys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn 85 90 95 ThrAla Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Ser Ala Tyr Tyr Val Asn Gln Asp Tyr Trp Gly Gln 115 120125 Gly Thr Ser Val Thr Val Ser Ser 130 135 34 base pairs nucleic acidsingle linear 30 GATAAGCTTC CACCATGGGC TTCAAGATGG AGTC 34 34 base pairsnucleic acid single linear 31 GGCGGATCCA CTCACGTTTG ATTTCCAGTT TGGT 3443 base pairs nucleic acid single linear 32 GATAAGCTTC CACCATGAAATGCAGCTGGG TCATGTTCTT CCT 43 34 base pairs nucleic acid single linear 33GGCGGATCCA CTCACCTGAG GAGACGGTGA CTGA 34 18 base pairs nucleic acidsingle linear 34 CAGACAGTGG TTCAAAGT 18 26 base pairs nucleic acidsingle linear 35 GAATTCGGAT CCACTCACGT TTGATT 26 44 base pairs nucleicacid single linear 36 AGTCAGAATG TGGGTACTAA TGTAGCCTGG TACCAGCAGA AGCC44 38 base pairs nucleic acid single linear 37 TCCTATCGGT ACAGTGGTGTGCCAAGCAGA TTCAGCGG 38 47 base pairs nucleic acid single linear 38GCTACCTACT ACTGCCAGCA ATATAACAGC TATCCTCGGG CGTTCGG 47 44 base pairsnucleic acid single linear 39 ACATTAGTAC CCACATTCTG ACTGGCCTTACAGGTGATGG TCAC 44 47 base pairs nucleic acid single linear 40GGCACACCAC TGTACCGATA GGATGCCGAG TAGATCAGCA GCTTTGG 47 44 base pairsnucleic acid single linear 41 GGATAGCTGT TATATTGCTG GCAGTAGTAGGTAGCGATGT CCTC 44 379 base pairs nucleic acid double linear CDS 1..378mat_peptide 58..378 42 ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCAACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala ThrAla Thr Gly -19 -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCAAGC AGC CTG AGC GCC 96 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser Ala 1 5 10 AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCCAGT CAG AAT GTG 144 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala SerGln Asn Val 15 20 25 GGT ACT AAT GTA GCC TGG TAC CAG CAG AAG CCA GGA AAGGCT CCA AAG 192 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys 30 35 40 45 CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT GGT GTGCCA AGC AGA 240 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val ProSer Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTC ACC ATCAGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile SerSer 65 70 75 CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGC CAG CAA TAT AACAGC 336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser80 85 90 TAT CCT CGG GCG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA 378 TyrPro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 C 379 126amino acids amino acid linear protein 43 Met Gly Trp Ser Cys Ile Ile LeuSer Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val His Ser Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 Gly Thr Asn Val Ala TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 Leu Leu Ile Tyr SerAla Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser GlySer Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu AspIle Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro Arg Ala PheGly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 31 base pairs nucleicacid single linear 44 GGTACCGACT ACACCTTCAC CATCAGCAGC C 31 31 basepairs nucleic acid single linear 45 GGTGAAGGTG TAGTCGGTAC CGCTACCGCT A31 379 base pairs nucleic acid double linear CDS 1..378 mat_peptide58..378 46 ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACAGGT 48 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly-19 -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTGAGC GCC 96 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla 1 5 10 AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCC AGT CAG AATGTG 144 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val15 20 25 GGT ACT AAT GTA GCC TGG TAC CAG CAG AAG CCA GGA AAG GCT CCA AAG192 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 3035 40 45 CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT GGT GTG CCA AGC AGA240 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 5055 60 TTC AGC GGT AGC GGT AGT GGT ACC GAC TAC ACC TTC ACC ATC AGC AGC288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser 6570 75 CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGC CAG CAA TAT AAC AGC336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 8085 90 TAT CCT CGG GCG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA 378 TyrPro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 C 379 126amino acids amino acid linear protein 47 Met Gly Trp Ser Cys Ile Ile LeuSer Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val His Ser Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 Gly Thr Asn Val Ala TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 Leu Leu Ile Tyr SerAla Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser GlySer Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu AspIle Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro Arg Ala PheGly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 29 base pairs nucleicacid single linear 48 GCTACCTACT TCTGCCAGCA ATATAACAG 29 29 base pairsnucleic acid single linear 49 TGCTGGCAGA AGTAGGTAGC GATGTCCTC 29 379base pairs nucleic acid double linear CDS 1..378 mat_peptide 58..378 50ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACA GGT 48 MetGly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15 -10-5 GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC 96Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCC AGT CAG AAT GTG 144 SerVal Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 GGTACT AAT GTA GCC TGG TAC CAG CAG AAG CCA GGA AAG GCT CCA AAG 192 Gly ThrAsn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 CTGCTG ATC TAC TCG GCA TCC TAT CGG TAC AGT GGT GTG CCA AGC AGA 240 Leu LeuIle Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 TTC AGCGGT AGC GGT AGC GGT ACC GAC TTC ACC TTC ACC ATC AGC AGC 288 Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 CTC CAG CCAGAG GAC ATC GCC ACC TAC TTC TGC CAG CAA TAT AAC AGC 336 Leu Gln Pro GluAsp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Asn Ser 80 85 90 TAT CCT CGG GCGTTC GGC CAA GGG ACC AAG GTG GAA ATC AAA 378 Tyr Pro Arg Ala Phe Gly GlnGly Thr Lys Val Glu Ile Lys 95 100 105 C 379 126 amino acids amino acidlinear protein 51 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala ThrAla Thr Gly -19 -15 -10 -5 Val His Ser Asp Ile Gln Met Thr Gln Ser ProSer Ser Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg Val Thr Ile Thr Cys LysAla Ser Gln Asn Val 15 20 25 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys ProGly Lys Ala Pro Lys 30 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg TyrSer Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr Phe Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu Asp Ile Ala Thr Tyr PheCys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro Arg Ala Phe Gly Gln Gly Thr LysVal Glu Ile Lys 95 100 105 379 base pairs nucleic acid double linear CDS1..378 mat_peptide 58..378 52 ATG GGA TGG AGC TGT ATC ATC CTC TCC TTGGTA GCA ACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu ValAla Thr Ala Thr Gly -19 -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAGAGC CCA AGC AGC CTG AGC GCC 96 Val His Ser Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala 1 5 10 AGC GTG GGT GAC AGA GTG ACC ATC ACC TGTAAG GCC AGT CAG AAT GTG 144 Ser Val Gly Asp Arg Val Thr Ile Thr Cys LysAla Ser Gln Asn Val 15 20 25 GGT ACT AAT GTA GCC TGG TAC CAG CAG AAG CCAGGA AAG GCT CCA AAG 192 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys 30 35 40 45 CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGTGGT GTG CCA AGC AGA 240 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser GlyVal Pro Ser Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TAC ACC TTCACC ATC AGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe ThrIle Ser Ser 65 70 75 CTC CAG CCA GAG GAC ATC GCC ACC TAC TTC TGC CAG CAATAT AAC AGC 336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln TyrAsn Ser 80 85 90 TAT CCT CGG GCG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA378 Tyr Pro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 C379 126 amino acids amino acid linear protein 53 Met Gly Trp Ser Cys IleIle Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val His Ser AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 Ser Val Gly AspArg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 Gly Thr Asn ValAla Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 Leu Leu IleTyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 Phe Ser GlySer Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser 65 70 75 Leu Gln ProGlu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro ArgAla Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 29 base pairsnucleic acid single linear 54 TGACAGAGTG TCCGTCACCT GTAAGGCCA 29 29 basepairs nucleic acid single linear 55 TTACAGGTGA CGGACACTCT GTCACCCAC 29379 base pairs nucleic acid double linear CDS 1..378 mat_peptide 58..37856 ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACA GGT 48Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15-10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC96 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 510 AGC GTG GGT GAC AGA GTG TCC GTC ACC TGT AAG GCC AGT CAG AAT GTG 144Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val 15 20 25GGT ACT AAT GTA GCC TGG TAC CAG CAG AAG CCA GGA AAG GCT CCA AAG 192 GlyThr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT GGT GTG CCA AGC AGA 240 LeuLeu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 TTCAGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTC ACC ATC AGC AGC 288 Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 CTC CAGCCA GAG GAC ATC GCC ACC TAC TAC TGC CAG CAA TAT AAC AGC 336 Leu Gln ProGlu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 80 85 90 TAT CCT CGGGCG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA 378 Tyr Pro Arg Ala Phe GlyGln Gly Thr Lys Val Glu Ile Lys 95 100 105 C 379 126 amino acids aminoacid linear protein 57 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val AlaThr Ala Thr Gly -19 -15 -10 -5 Val His Ser Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg Val Ser Val Thr CysLys Ala Ser Gln Asn Val 15 20 25 Gly Thr Asn Val Ala Trp Tyr Gln Gln LysPro Gly Lys Ala Pro Lys 30 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr ArgTyr Ser Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Phe Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu Asp Ile Ala Thr TyrTyr Cys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro Arg Ala Phe Gly Gln Gly ThrLys Val Glu Ile Lys 95 100 105 379 base pairs nucleic acid double linearCDS 1..378 mat_peptide 58..378 58 ATG GGA TGG AGC TGT ATC ATC CTC TCCTTG GTA GCA ACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu Ser LeuVal Ala Thr Ala Thr Gly -19 -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACCCAG AGC CCA AGC AGC CTG AGC GCC 96 Val His Ser Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu Ser Ala 1 5 10 AGC GTG GGT GAC AGA GTG TCC GTC ACCTGT AAG GCC AGT CAG AAT GTG 144 Ser Val Gly Asp Arg Val Ser Val Thr CysLys Ala Ser Gln Asn Val 15 20 25 GGT ACT AAT GTA GCC TGG TAC CAG CAG AAGCCA GGA AAG GCT CCA AAG 192 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys ProGly Lys Ala Pro Lys 30 35 40 45 CTG CTG ATC TAC TCG GCA TCC TAT CGG TACAGT GGT GTG CCA AGC AGA 240 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr SerGly Val Pro Ser Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACCTTC ACC ATC AGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr PheThr Ile Ser Ser 65 70 75 CTC CAG CCA GAG GAC ATC GCC ACC TAC TTC TGC CAGCAA TAT AAC AGC 336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln GlnTyr Asn Ser 80 85 90 TAT CCT CGG GCG TTC GGC CAA GGG ACC AAG GTG GAA ATCAAA 378 Tyr Pro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100105 C 379 126 amino acids amino acid linear protein 59 Met Gly Trp SerCys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val HisSer Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 Ser ValGly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 Gly ThrAsn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 LeuLeu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 PheSer Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 LeuGln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Asn Ser 80 85 90 TyrPro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 26 basepairs nucleic acid single linear 60 GACTTCACCT TGACCATCAG CAGCCT 26 26base pairs nucleic acid single linear 61 CTGCTGATGG TCAAGGTGAA GTCGGT 26379 base pairs nucleic acid double linear CDS 1..378 mat_peptide 58..37862 ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACA GGT 48Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15-10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC96 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 510 AGC GTG GGT GAC AGA GTG TCC GTC ACC TGT AAG GCC AGT CAG AAT GTG 144Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val 15 20 25GGT ACT AAT GTA GCC TGG TAC CAG CAG AAG CCA GGA AAG GCT CCA AAG 192 GlyThr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT GGT GTG CCA AGC AGA 240 LeuLeu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 TTCAGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTG ACC ATC AGC AGC 288 Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 CTC CAGCCA GAG GAC ATC GCC ACC TAC TTC TGC CAG CAA TAT AAC AGC 336 Leu Gln ProGlu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Asn Ser 80 85 90 TAT CCT CGGGCG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA 378 Tyr Pro Arg Ala Phe GlyGln Gly Thr Lys Val Glu Ile Lys 95 100 105 C 379 126 amino acids aminoacid linear protein 63 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val AlaThr Ala Thr Gly -19 -15 -10 -5 Val His Ser Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg Val Ser Val Thr CysLys Ala Ser Gln Asn Val 15 20 25 Gly Thr Asn Val Ala Trp Tyr Gln Gln LysPro Gly Lys Ala Pro Lys 30 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr ArgTyr Ser Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu Asp Ile Ala Thr TyrPhe Cys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro Arg Ala Phe Gly Gln Gly ThrLys Val Glu Ile Lys 95 100 105 379 base pairs nucleic acid double linearCDS 1..378 mat_peptide 58..378 64 ATG GGA TGG AGC TGT ATC ATC CTC TCCTTG GTA GCA ACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu Ser LeuVal Ala Thr Ala Thr Gly -19 -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACCCAG AGC CCA AGC AGC CTG AGC GCC 96 Val His Ser Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu Ser Ala 1 5 10 AGC GTG GGT GAC AGA GTG ACC ATC ACCTGT AAG GCC AGT CAG AAT GTG 144 Ser Val Gly Asp Arg Val Thr Ile Thr CysLys Ala Ser Gln Asn Val 15 20 25 GGT ACT AAT GTA GCC TGG TAC CAG CAG AAGCCA GGA AAG GCT CCA AAG 192 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys ProGly Lys Ala Pro Lys 30 35 40 45 CTG CTG ATC TAC TCG GCA TCC TAT CGG TACAGT GGT GTG CCA AGC AGA 240 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr SerGly Val Pro Ser Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACCTTG ACC ATC AGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuThr Ile Ser Ser 65 70 75 CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGC CAGCAA TAT AAC AGC 336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln GlnTyr Asn Ser 80 85 90 TAT CCT CGG GCG TTC GGC CAA GGG ACC AAG GTG GAA ATCAAA 378 Tyr Pro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100105 C 379 126 amino acids amino acid linear protein 65 Met Gly Trp SerCys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val HisSer Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 Ser ValGly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 Gly ThrAsn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 LeuLeu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 PheSer Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 LeuGln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 80 85 90 TyrPro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 29 basepairs nucleic acid single linear 66 GGACAGAGTC CAAAGCCGCT GATCTACTC 2929 base pairs nucleic acid single linear 67 ATCAGCGGCT TTGGACTCTGTCCTGGCTT 29 379 base pairs nucleic acid double linear CDS 1..378mat_peptide 58..378 68 ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCAACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala ThrAla Thr Gly -19 -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCAAGC AGC CTG AGC GCC 96 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser Ala 1 5 10 AGC GTG GGT GAC AGA GTG TCC GTC ACC TGT AAG GCCAGT CAG AAT GTG 144 Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala SerGln Asn Val 15 20 25 GGT ACT AAT GTA GCC TGG TAC CAG CAG AAG CCA GGA CAGAGT CCA AAG 192 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln SerPro Lys 30 35 40 45 CCG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT GGT GTGCCA AGC AGA 240 Pro Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val ProSer Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTG ACC ATCAGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer 65 70 75 CTC CAG CCA GAG GAC ATC GCC ACC TAC TTC TGC CAG CAA TAT AACAGC 336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Asn Ser80 85 90 TAT CCT CGG GCG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA 378 TyrPro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 C 379 126amino acids amino acid linear protein 69 Met Gly Trp Ser Cys Ile Ile LeuSer Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val His Ser Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg ValSer Val Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 Gly Thr Asn Val Ala TrpTyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 30 35 40 45 Pro Leu Ile Tyr SerAla Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser GlySer Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu AspIle Ala Thr Tyr Phe Cys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro Arg Ala PheGly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 26 base pairs nucleicacid single linear 70 GAGGACATCG CTGACTACTT CTGCCA 26 26 base pairsnucleic acid single linear 71 AAGTAGTCAG CGATGTCCTC TGGCTG 26 379 basepairs nucleic acid double linear CDS 1..378 mat_peptide 58..378 72 ATGGGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACA GGT 48 Met GlyTrp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC 96 ValHis Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 AGCGTG GGT GAC AGA GTG TCC GTC ACC TGT AAG GCC AGT CAG AAT GTG 144 Ser ValGly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 GGT ACTAAT GTA GCC TGG TAC CAG CAG AAG CCA GGA CAG AGT CCA AAG 192 Gly Thr AsnVal Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 30 35 40 45 CCG CTGATC TAC TCG GCA TCC TAT CGG TAC AGT GGT GTG CCA AGC AGA 240 Pro Leu IleTyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 TTC AGC GGTAGC GGT AGC GGT ACC GAC TTC ACC TTG ACC ATC AGC AGC 288 Phe Ser Gly SerGly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 CTC CAG CCA GAGGAC ATC GCC GAC TAC TTC TGC CAG CAA TAT AAC AGC 336 Leu Gln Pro Glu AspIle Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser 80 85 90 TAT CCT CGG GCG TTCGGC CAA GGG ACC AAG GTG GAA ATC AAA 378 Tyr Pro Arg Ala Phe Gly Gln GlyThr Lys Val Glu Ile Lys 95 100 105 C 379 126 amino acids amino acidlinear protein 73 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala ThrAla Thr Gly -19 -15 -10 -5 Val His Ser Asp Ile Gln Met Thr Gln Ser ProSer Ser Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg Val Ser Val Thr Cys LysAla Ser Gln Asn Val 15 20 25 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys ProGly Gln Ser Pro Lys 30 35 40 45 Pro Leu Ile Tyr Ser Ala Ser Tyr Arg TyrSer Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr Leu Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu Asp Ile Ala Asp Tyr PheCys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro Arg Ala Phe Gly Gln Gly Thr LysVal Glu Ile Lys 95 100 105 379 base pairs nucleic acid double linear CDS1..378 mat_peptide 58..378 74 ATG GGA TGG AGC TGT ATC ATC CTC TCC TTGGTA GCA ACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu ValAla Thr Ala Thr Gly -19 -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAGAGC CCA AGC AGC CTG AGC GCC 96 Val His Ser Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala 1 5 10 AGC GTG GGT GAC AGA GTG TCC GTC ACC TGTAAG GCC AGT CAG AAT GTG 144 Ser Val Gly Asp Arg Val Ser Val Thr Cys LysAla Ser Gln Asn Val 15 20 25 GGT ACT AAT GTA GCC TGG TAC CAG CAG AAG CCAGGA AAG GCT CCA AAG 192 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys 30 35 40 45 CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGTGGT GTG CCA AGC AGA 240 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser GlyVal Pro Ser Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTGACC ATC AGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu ThrIle Ser Ser 65 70 75 CTC CAG CCA GAG GAC ATC GCC GAC TAC TTC TGC CAG CAATAT AAC AGC 336 Leu Gln Pro Glu Asp Ile Ala Asp Tyr Phe Cys Gln Gln TyrAsn Ser 80 85 90 TAT CCT CGG GCG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA378 Tyr Pro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 C379 126 amino acids amino acid linear protein 75 Met Gly Trp Ser Cys IleIle Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val His Ser AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 Ser Val Gly AspArg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 Gly Thr Asn ValAla Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 Leu Leu IleTyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 Leu Gln ProGlu Asp Ile Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro ArgAla Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 379 base pairsnucleic acid double linear CDS 1..378 mat_peptide 58..378 76 ATG GGA TGGAGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACA GGT 48 Met Gly Trp SerCys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 GTC CACTCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC 96 Val His SerAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 AGC GTG GGTGAC AGA GTG TCC GTC ACC TGT AAG GCC AGT CAG AAT GTG 144 Ser Val Gly AspArg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 GGT ACT AAT GTAGCC TGG TAC CAG CAG AAG CCA GGA CAG AGT CCA AAG 192 Gly Thr Asn Val AlaTrp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 30 35 40 45 CCG CTG ATC TACTCG GCA TCC TAT CGG TAC AGT GGT GTG CCA AGC AGA 240 Pro Leu Ile Tyr SerAla Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 TTC AGC GGT AGC GGTAGC GGT ACC GAC TTC ACC TTC ACC ATC AGC AGC 288 Phe Ser Gly Ser Gly SerGly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 CTC CAG CCA GAG GAC ATCGCC ACC TAC TAC TGC CAG CAA TAT AAC AGC 336 Leu Gln Pro Glu Asp Ile AlaThr Tyr Tyr Cys Gln Gln Tyr Asn Ser 80 85 90 TAT CCT CGG GCG TTC GGC CAAGGG ACC AAG GTG GAA ATC AAA 378 Tyr Pro Arg Ala Phe Gly Gln Gly Thr LysVal Glu Ile Lys 95 100 105 C 379 126 amino acids amino acid linearprotein 77 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr Ala ThrGly -19 -15 -10 -5 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser Ala 1 5 10 Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala SerGln Asn Val 15 20 25 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly GlnSer Pro Lys 30 35 40 45 Pro Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser GlyVal Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr PheThr Ile Ser Ser 65 70 75 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys GlnGln Tyr Asn Ser 80 85 90 Tyr Pro Arg Ala Phe Gly Gln Gly Thr Lys Val GluIle Lys 95 100 105 26 base pairs nucleic acid single linear 78CAGAGCCAAA AGTTCCTGAG CGCCAG 26 26 base pairs nucleic acid single linear79 CTCAGGAACT TTTGGCTCTG GGTCAT 26 379 base pairs nucleic acid doublelinear CDS 1..378 mat_peptide 58..378 80 ATG GGA TGG AGC TGT ATC ATC CTCTCC TTG GTA GCA ACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu SerLeu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 GTC CAC TCC GAC ATC CAG ATGACC CAG AGC CAA AAG TTC CTG AGC GCC 96 Val His Ser Asp Ile Gln Met ThrGln Ser Gln Lys Phe Leu Ser Ala 1 5 10 AGC GTG GGT GAC AGA GTG ACC ATCACC TGT AAG GCC AGT CAG AAT GTG 144 Ser Val Gly Asp Arg Val Thr Ile ThrCys Lys Ala Ser Gln Asn Val 15 20 25 GGT ACT AAT GTA GCC TGG TAC CAG CAGAAG CCA GGA CAG AGT CCA AAG 192 Gly Thr Asn Val Ala Trp Tyr Gln Gln LysPro Gly Gln Ser Pro Lys 30 35 40 45 CCG CTG ATC TAC TCG GCA TCC TAT CGGTAC AGT GGT GTG CCA AGC AGA 240 Pro Leu Ile Tyr Ser Ala Ser Tyr Arg TyrSer Gly Val Pro Ser Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TTCACC TTC ACC ATC AGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrPhe Thr Ile Ser Ser 65 70 75 CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGCCAG CAA TAT AAC AGC 336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys GlnGln Tyr Asn Ser 80 85 90 TAT CCT CGG GCG TTC GGC CAA GGG ACC AAG GTG GAAATC AAA 378 Tyr Pro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95100 105 C 379 126 amino acids amino acid linear protein 81 Met Gly TrpSer Cys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 ValHis Ser Asp Ile Gln Met Thr Gln Ser Gln Lys Phe Leu Ser Ala 1 5 10 SerVal Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 GlyThr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 30 35 40 45Pro Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 80 85 90Tyr Pro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 29base pairs nucleic acid single linear 82 GGACAGAGTC CAAAGCTGCT GATCTACTC29 29 base pairs nucleic acid single linear 83 ATCAGCAGCT TTGGACTCTGTCCTGGCTT 29 379 base pairs nucleic acid double linear CDS 1..378mat_peptide 58..378 84 ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCAACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala ThrAla Thr Gly -19 -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCAAGC AGC CTG AGC GCC 96 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser Ala 1 5 10 AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCCAGT CAG AAT GTG 144 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala SerGln Asn Val 15 20 25 GGT ACT AAT GTA GCC TGG TAC CAG CAG AAG CCA GGA CAGAGT CCA AAG 192 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln SerPro Lys 30 35 40 45 CTG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT GGT GTGCCA AGC AGA 240 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val ProSer Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTC ACC ATCAGC AGC 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile SerSer 65 70 75 CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGC CAG CAA TAT AACAGC 336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser80 85 90 TAT CCT CGG GCG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA 378 TyrPro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 C 379 126amino acids amino acid linear protein 85 Met Gly Trp Ser Cys Ile Ile LeuSer Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val His Ser Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 Gly Thr Asn Val Ala TrpTyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 30 35 40 45 Leu Leu Ile Tyr SerAla Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser GlySer Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu AspIle Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro Arg Ala PheGly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 379 base pairs nucleicacid double linear CDS 1..378 mat_peptide 58..378 86 ATG GGA TGG AGC TGTATC ATC CTC TCC TTG GTA GCA ACA GCT ACA GGT 48 Met Gly Trp Ser Cys IleIle Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 GTC CAC TCC GACATC CAG ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC 96 Val His Ser Asp IleGln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 AGC GTG GGT GAC AGAGTG ACC ATC ACC TGT AAG GCC AGT CAG AAT GTG 144 Ser Val Gly Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 GGT ACT AAT GTA GCC TGGTAC CAG CAG AAG CCA GGA CAG AGT CCA AAG 192 Gly Thr Asn Val Ala Trp TyrGln Gln Lys Pro Gly Gln Ser Pro Lys 30 35 40 45 CCG CTG ATC TAC TCG GCATCC TAT CGG TAC AGT GGT GTG CCA AGC AGA 240 Pro Leu Ile Tyr Ser Ala SerTyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 TTC AGC GGT AGC GGT AGC GGTACC GAC TTC ACC TTC ACC ATC AGC AGC 288 Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Thr Phe Thr Ile Ser Ser 65 70 75 CTC CAG CCA GAG GAC ATC GCC ACCTAC TAC TGC CAG CAA TAT AAC AGC 336 Leu Gln Pro Glu Asp Ile Ala Thr TyrTyr Cys Gln Gln Tyr Asn Ser 80 85 90 TAT CCT CGG GCG TTC GGC CAA GGG ACCAAG GTG GAA ATC AAA 378 Tyr Pro Arg Ala Phe Gly Gln Gly Thr Lys Val GluIle Lys 95 100 105 C 379 126 amino acids amino acid linear protein 87Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly -19 -15-10 -5 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 15 10 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val 1520 25 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 3035 40 45 Pro Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser65 70 75 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser80 85 90 Tyr Pro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100105 23 base pairs nucleic acid single linear 88 GCTCCAAAGC CGCTGATCTACTC 23 23 base pairs nucleic acid single linear 89 TAGATCAGCG GCTTTGGAGCCTT 23 379 base pairs nucleic acid double linear CDS 1..378 mat_peptide58..378 90 ATG GGA TGG AGC TGT ATC ATC CTC TCC TTG GTA GCA ACA GCT ACAGGT 48 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Val Ala Thr Ala Thr Gly-19 -15 -10 -5 GTC CAC TCC GAC ATC CAG ATG ACC CAG AGC CCA AGC AGC CTGAGC GCC 96 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla 1 5 10 AGC GTG GGT GAC AGA GTG ACC ATC ACC TGT AAG GCC AGT CAG AATGTG 144 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val15 20 25 GGT ACT AAT GTA GCC TGG TAC CAG CAG AAG CCA GGA AAG GCT CCA AAG192 Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 3035 40 45 CCG CTG ATC TAC TCG GCA TCC TAT CGG TAC AGT GGT GTG CCA AGC AGA240 Pro Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 5055 60 TTC AGC GGT AGC GGT AGC GGT ACC GAC TTC ACC TTC ACC ATC AGC AGC288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 6570 75 CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGC CAG CAA TAT AAC AGC336 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 8085 90 TAT CCT CGG GCG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA 378 TyrPro Arg Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 C 379 126amino acids amino acid linear protein 91 Met Gly Trp Ser Cys Ile Ile LeuSer Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val His Ser Asp Ile GlnMet Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 Ser Val Gly Asp Arg ValThr Ile Thr Cys Lys Ala Ser Gln Asn Val 15 20 25 Gly Thr Asn Val Ala TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 30 35 40 45 Pro Leu Ile Tyr SerAla Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser GlySer Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 Leu Gln Pro Glu AspIle Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 80 85 90 Tyr Pro Arg Ala PheGly Gln Gly Thr Lys Val Glu Ile Lys 95 100 105 137 base pairs nucleicacid single linear 92 AAGAAGCCTG GGTCCTCAGT GAAGGTCTCC TGCAAGGCTTCTGGCTTCAA CATTAAAGAC 60 ACCTATATAC ACTGGGTGCG CCAGGCTCCA GGACAGGGCCTGGAGTGGAT GGGAAGGAT 120 GATCCTGAGG ATGGTAA 137 111 base pairs nucleicacid single linear 93 TGAGATCTGA GGACACAGCC TTTTATTTCT GTGCAAGTGCCTACTATGTT AACCAGGACT 60 ACTGGGGCCA AGGGACCACT GTCACCGTCT CCTCAGGTGAGTGGATCCGA C 111 130 base pairs nucleic acid single linear 94 ACCTTCACTGAGGACCCAGG CTTCTTCACC TCAGCTCCAG ACTGCACCAG CTGCACCTGG 60 GAGTGAGCACCTGGAGCTAC AGCCAGCAAG AAGAAGACCC TCCAGGTCCA GTCCATGGT 120 GAAGCTTATC 130132 base pairs nucleic acid single linear 95 AAAGGCTGTG TCCTCAGATCTCAGGCTGCT GAGCTCCATG TAGGCTGTGT TCGTGGATTC 60 GTCTGCAGTG ATTGTGACTCGGCCCTGGAA CTTCGGGTCA TATTTAGTAT TACCATCCG 120 AGGATCAATC CT 132 25 basepairs nucleic acid single linear 96 GATAAGCTTC CACCATGGAC TGGAC 25 25base pairs nucleic acid single linear 97 GTCGGATCCA CTCACCTGAG GAGAC 25409 base pairs nucleic acid double linear CDS 1..408 mat_peptide 58..40898 ATG GAC TGG ACC TGG AGG GTC TTC TTC TTG CTG GCT GTA GCT CCA GGT 48Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Ala Val Ala Pro Gly -19 -15-10 -5 GCT CAC TCC CAG GTG CAG CTG GTG CAG TCT GGA GCT GAG GTG AAG AAG96 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 510 CCT GGG TCC TCA GTG AAG GTC TCC TGC AAG GCT TCT GGC TTC AAC ATT 144Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25AAA GAC ACC TAT ATA CAC TGG GTG CGC CAG GCT CCA GGA CAG GGC CTG 192 LysAsp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45GAG TGG ATG GGA AGG ATT GAT CCT GCG GAT GGT AAT ACT AAA TAT GAC 240 GluTrp Met Gly Arg Ile Asp Pro Ala Asp Gly Asn Thr Lys Tyr Asp 50 55 60 CCGAAG TTC CAG GGC CGA GTC ACA ATC ACT GCA GAC GAA TCC ACG AAC 288 Pro LysPhe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn 65 70 75 ACA GCCTAC ATG GAG CTC AGC AGC CTG AGA TCT GAG GAC ACA GCC TTT 336 Thr Ala TyrMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe 80 85 90 TAT TTC TGTGCA AGT GCC TAC TAT GTT AAC CAG GAC TAC TGG GGC CAA 384 Tyr Phe Cys AlaSer Ala Tyr Tyr Val Asn Gln Asp Tyr Trp Gly Gln 95 100 105 GGG ACC ACTGTC ACC GTC TCC TCA G 409 Gly Thr Thr Val Thr Val Ser Ser 110 115 136amino acids amino acid linear protein 99 Met Asp Trp Thr Trp Arg Val PhePhe Leu Leu Ala Val Ala Pro Gly -19 -15 -10 -5 Ala His Ser Gln Val GlnLeu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 Pro Gly Ser Ser Val LysVal Ser Cys Lys Ala Ser Gly Phe Asn Ile 15 20 25 Lys Asp Thr Tyr Ile HisTrp Val Arg Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly ArgIle Asp Pro Ala Asp Gly Asn Thr Lys Tyr Asp 50 55 60 Pro Lys Phe Gln GlyArg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn 65 70 75 Thr Ala Tyr Met GluLeu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe 80 85 90 Tyr Phe Cys Ala SerAla Tyr Tyr Val Asn Gln Asp Tyr Trp Gly Gln 95 100 105 Gly Thr Thr ValThr Val Ser Ser 110 115 84 base pairs nucleic acid single linear 100AGCTTGTCAC CGTCTCCTCA GGTGGTGGTG GTTCGGGTGG TGGTGGTTCG GGTGGTGGCG 60GATCGGACAT CCAGATGACC CAGG 84 84 base pairs nucleic acid single linear101 AATTCCTGGG CCATCTGGAT GTCCGATCCG CCACCACCCG AACCACCACC ACCCGAACCA 60CCACCACCTG AGGAGACGGT GACA 84 34 base pairs nucleic acid single linear102 CAGCCATGGC GCAGTGTGCA GCTGGTGCAG TCTG 34 41 base pairs nucleic acidsingle linear 103 CCACCCGAAC CACCACCACC TGAGGAGACG GTGACAGTGG T 41 41base pairs nucleic acid single linear 104 GGGACCACTG TCACCGTCTCCTCAGGTGGT GGTGGTTCGG G 41 41 base pairs nucleic acid single linear 105GGGCTCTGGG TCATCTGGAT GTCCGATCCG CCACCACCCG A 41 44 base pairs nucleicacid single linear 106 TCGGACATCC AGATGACCCA GAGCCCAAGC AGCCTGAGCG CCAG44 57 base pairs nucleic acid single linear 107 CAAGAATTCT TATTATTTATCGTCATCGTC TTTGTAGTCT TTGATTTCGA CCTTGGT 57 822 base pairs nucleic aciddouble linear CDS 1..807 mat_peptide 1..807 108 ATG AAA TAC CTA TTG CCTACG GCA GCC GCT GGA TTG TTA TTA CTC GCT 48 Met Lys Tyr Leu Leu Pro ThrAla Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 GCC CAA CCA GCC ATG GCGCAG GTG CAG CTG GTG CAG TCT GGA GCT GAG 96 Ala Gln Pro Ala Met Ala GlnVal Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 GTG AAG AAG CCT GGG TCC TCAGTG AAG GTC TCC TGC AAG GCT TCT GGC 144 Val Lys Lys Pro Gly Ser Ser ValLys Val Ser Cys Lys Ala Ser Gly 35 40 45 TTC AAC ATT AAA GAC ACC TAT ATACAC TGG GTG CGC CAG GCT CCA GGA 192 Phe Asn Ile Lys Asp Thr Tyr Ile HisTrp Val Arg Gln Ala Pro Gly 50 55 60 CAG GGC CTG GAG TGG ATG GGA AGG ATTGAT CCT GCG GAT GGT AAT ACT 240 Gln Gly Leu Glu Trp Met Gly Arg Ile AspPro Ala Asp Gly Asn Thr 65 70 75 80 AAA TAT GAC CCG AAG TTC CAG GGC CGAGTC ACA ATC ACT GCA GAC GAA 288 Lys Tyr Asp Pro Lys Phe Gln Gly Arg ValThr Ile Thr Ala Asp Glu 85 90 95 TCC ACG AAC ACA GCC TAC ATG GAG CTC AGCAGC CTG AGA TCT GAG GAC 336 Ser Thr Asn Thr Ala Tyr Met Glu Leu Ser SerLeu Arg Ser Glu Asp 100 105 110 ACA GCC TTT TAT TTC TGT GCA AGT GCC TACTAT GTT AAC CAG GAC TAC 384 Thr Ala Phe Tyr Phe Cys Ala Ser Ala Tyr TyrVal Asn Gln Asp Tyr 115 120 125 TGG GGC CAA GGG ACC ACT GTC ACC GTC TCCTCA GGT GGT GGT GGT TCG 432 Trp Gly Gln Gly Thr Thr Val Thr Val Ser SerGly Gly Gly Gly Ser 130 135 140 GGT GGT GGT GGT TCG GGT GGT GGC GGA TCGGAC ATC CAG ATG ACC CAG 480 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser AspIle Gln Met Thr Gln 145 150 155 160 AGC CCA AGC AGC CTG AGC GCC AGC GTGGGT GAC AGA GTG ACC ATC ACC 528 Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Arg Val Thr Ile Thr 165 170 175 TGT AAG GCC AGT CAG AAT GTG GGT ACTAAT GTA GCC TGG TAC CAG CAG 576 Cys Lys Ala Ser Gln Asn Val Gly Thr AsnVal Ala Trp Tyr Gln Gln 180 185 190 AAG CCA GGA AAG GCT CCA AAG CCG CTGATC TAC TCG GCA TCC TAT CGG 624 Lys Pro Gly Lys Ala Pro Lys Pro Leu IleTyr Ser Ala Ser Tyr Arg 195 200 205 TAC AGT GGT GTG CCA AGC AGA TTC AGCGGT AGC GGT AGC GGT ACC GAC 672 Tyr Ser Gly Val Pro Ser Arg Phe Ser GlySer Gly Ser Gly Thr Asp 210 215 220 TTC ACC TTC ACC ATC AGC AGC CTC CAGCCA GAG GAC ATC GCT ACC TAC 720 Phe Thr Phe Thr Ile Ser Ser Leu Gln ProGlu Asp Ile Ala Thr Tyr 225 230 235 240 TAC TGC CAG CAA TAT AAC AGC TATCCT CGG GCG TTC GGC CAA GGG ACC 768 Tyr Cys Gln Gln Tyr Asn Ser Tyr ProArg Ala Phe Gly Gln Gly Thr 245 250 255 AAG GTC GAA ATC AAA GAC TAC AAAGAC GAT GAC GAT AAA TAATAAGAAT 817 Lys Val Glu Ile Lys Asp Tyr Lys AspAsp Asp Asp Lys 260 265 TCTTG 822 269 amino acids amino acid linearprotein 109 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu LeuAla 1 5 10 15 Ala Gln Pro Ala Met Ala Gln Val Gln Leu Val Gln Ser GlyAla Glu 20 25 30 Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys AlaSer Gly 35 40 45 Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln AlaPro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Ala Asp GlyAsn Thr 65 70 75 80 Lys Tyr Asp Pro Lys Phe Gln Gly Arg Val Thr Ile ThrAla Asp Glu 85 90 95 Ser Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu ArgSer Glu Asp 100 105 110 Thr Ala Phe Tyr Phe Cys Ala Ser Ala Tyr Tyr ValAsn Gln Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Thr Val Thr Val Ser SerGly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly SerAsp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala SerVal Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Lys Ala Ser Gln Asn ValGly Thr Asn Val Ala Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala ProLys Pro Leu Ile Tyr Ser Ala Ser Tyr Arg 195 200 205 Tyr Ser Gly Val ProSer Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Phe ThrIle Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr 225 230 235 240 Tyr CysGln Gln Tyr Asn Ser Tyr Pro Arg Ala Phe Gly Gln Gly Thr 245 250 255 LysVal Glu Ile Lys Asp Tyr Lys Asp Asp Asp Asp Lys 260 265 45 base pairsnucleic acid double linear CDS 1..45 mat_peptide 1..45 110 GGT GGT GGTGGT TCG GGT GGT GGT GGT TCG GGT GGT GGC GGA TCG 45 Gly Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 15 amino acids aminoacid linear protein 111 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly GlyGly Gly Ser 1 5 10 15 87 amino acids amino acid linear protein 112 AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gl 1 5 10 15 AspArg Val Thr Ile Thr Cys Trp Tyr Gln Gln Lys Pro Gly Lys Al 20 25 30 ProLys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pr 35 40 45 SerArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Il 50 55 60 SerSer Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Phe Gly Gl 65 70 75 80Gly Thr Lys Val Glu Ile Lys 85 76 amino acids amino acid linear protein113 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Se 1 510 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Trp Va 2025 30 Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Val Thr Il 3540 45 Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Le 5055 60 Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys Ala Gly 65 70 75 11 aminoacids amino acid linear protein 114 Trp Gly Gln Gly Thr Thr Val Thr ValSer Ser 1 5 10 5 amino acids amino acid linear peptide 115 Asp Thr TyrIle His 1 5 17 amino acids amino acid linear protein 116 Arg Ile Asp ProAla Asp Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gl 1 5 10 15 Gly 8 aminoacids amino acid linear peptide 117 Ala Tyr Tyr Val Asn Gln Asp Tyr 1 511 amino acids amino acid linear protein 118 Lys Ala Ser Gln Asn Val GlyThr Asn Val Ala 1 5 10 7 amino acids amino acid linear peptide 119 SerAla Ser Tyr Arg Tyr Ser 1 5 9 amino acids amino acid linear peptide 120Gln Gln Tyr Asn Ser Tyr Pro Arg Ala 1 5 23 amino acids amino acid linearprotein 121 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValGl 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 15 amino acids amino acidlinear protein 122 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro LeuIle Tyr 1 5 10 15 32 amino acids amino acid linear protein 123 Gly ValPro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Th 1 5 10 15 Phe ThrIle Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cy 20 25 30 10 aminoacids amino acid linear peptide 124 Phe Gly Gln Gly Thr Lys Val Glu IleLys 1 5 10 15 amino acids amino acid linear protein 125 Trp Tyr Gln GlnLys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr 1 5 10 15 30 amino acidsamino acid linear protein 126 Gln Val Gln Leu Val Gln Ser Gly Ala GluVal Lys Lys Pro Gly Se 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser GlyPhe Asn Ile Lys 20 25 30 14 amino acids amino acid linear protein 127Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 32 aminoacids amino acid linear protein 128 Arg Val Thr Ile Thr Ala Asp Glu SerThr Asn Thr Ala Tyr Met Gl 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp ThrAla Phe Tyr Phe Cys Ala Se 20 25 30 11 amino acids amino acid linearpeptide 129 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 107 aminoacids amino acid linear protein 130 Asp Ile Gln Met Thr Gln Ser Pro SerSer Leu Ser Ala Ser Val Gl 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys AlaSer Gln Asn Val Gly Thr As 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys Pro Leu Il 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser GlyVal Pro Ser Arg Phe Ser Gl 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr PheThr Ile Ser Ser Leu Gln Pr 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr CysGln Gln Tyr Asn Ser Tyr Pro Ar 85 90 95 Ala Phe Gly Gln Gly Thr Lys ValGlu Ile Lys 100 105 107 amino acids amino acid linear protein 131 AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gl 1 5 10 15 AspArg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr As 20 25 30 ValAla Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Il 35 40 45 TyrSer Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gl 50 55 60 SerGly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pr 65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Ar 85 90 95Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 117 amino acidsamino acid linear protein 132 Gln Val Gln Leu Val Gln Ser Gly Ala GluVal Lys Lys Pro Gly Se 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser GlyPhe Asn Ile Lys Asp Th 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro GlyGln Gly Leu Glu Trp Me 35 40 45 Gly Arg Ile Asp Pro Ala Asp Gly Asn ThrLys Tyr Asp Pro Lys Ph 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp GluSer Thr Asn Thr Ala Ty 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser GluAsp Thr Ala Phe Tyr Phe Cy 85 90 95 Ala Ser Ala Tyr Tyr Val Asn Gln AspTyr Trp Gly Gln Gly Thr Th 100 105 110 Val Thr Val Ser Ser 115

1. An L chain variable region (V region) of an antibody to humanmedulloblastoma cells, comprising three complementarity determiningregions (CDRs) having the amino acid sequences defined below: CDR1: LysAla Ser Gln Asn Val Gly Thr Asn Val Ala CDR2: Ser Ala Ser Tyr Arg TyrSer CDR3; Gln Gln Tyr Asn Ser Tyr Pro Arg Ala or a portion thereof andfour framework regions (FRs).
 2. An L chain of antibody to humanmedulloblastoma cells comprising the L chain variable region (V region)of claim I and human L chain constant region (C region).
 3. An L chainaccording to claim 2 wherein the FRs of said L chain V region arederived from a mouse antibody.
 4. An L chain according to claim 2wherein said L chain V region has the amino acid sequence indicated inSEQ ID NO:26.
 5. An L chain according to claim 2 wherein the FRs of saidL chain V region are derived from a human antibody.
 6. An L chainaccording to claim 2 or 5 wherein the FRs of said L chain V region arederived from a human antibody REI.
 7. An L chain according to claim 5 or6 wherein the amino acid at position 46 in the second FR of said L chainV region is praline.
 8. An L chain according to claim 5 or 6 wherein theamino acids at positions 42, 43 and 46 in the second FR of said L chainV region are glutamine, serine and proline, respectively.
 9. An L chainaccording to claim 2 wherein said L chain V region includes either setof four FRs having the following amino acid sequences: (1) FRI: Asp IleGln met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val ThrIle Thr Cys FR2: Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu IleTyr FR3: Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrPhe Thr Ile Ser Ser reu Gln Pro Clu Asp lie Ala Txr Tyr Tyr Cys FR4: PheGly Gln Gly Thr Lys Val Glu Ile Lys (2) FR1: Asp Ile Gln Met Thr Gln SerPro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys FR2: TrpTyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr FR3: Gly Val ProSer Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser SerLeu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys FR4: Phe Gly Gln Cly Thr LysVal Glu Ile Lys
 10. An L chain according to claim 2 wherein said human Lchain C region is a KC region.
 11. An H chain V region of an antibody tohuman medulloblastoma cells containing three CDRs having the amino acidsequences defined below: CDR1; Asp Thr Tyr Ile His CDR2: Arg Ile Asp ProAla Asp Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly CDR3: Ala Tyr TyrVal Asn Gln Asp Tyr or a portion thereof and four FRs.
 12. An H chain ofantibody to human medulloblastoma cells comprising the H chain V regionof claim 11 and a human H C region.
 13. An H chain according to claim 12wherein the FRs of said H chain V region are derived from a mouseantibody.
 14. The H chain according to claim 12 wherein said H chain Vregion has the amino acid sequence indicated in SEQ ID NO:27.
 15. An Hchain according to claim 12 wherein the FRs of said H chain V region arederived from a human antibody.
 16. An H chain according to claim 12 or15 wherein the FRs of said H chain V region are derived from a humanantibody of subgroup I.
 17. An H chain according to claim 12 or 15wherein the FRs of said H chain V region are derived from a humanantibody Eu.
 18. The H chain according to claim 15 wherein said H chainV region contains four FRs having the following amino acid sequences;FR1: Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser SerVal Lys Val Ser Ws Lys Ala Ser Gly Phe Asn Ile Lys FR2: Trp Val Arg GlnAla Pro Gly Gln Gly Leu Glu Trp Met Gly FR3: Arg Val Thr Ile Thr Ala AspGlu Ser Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser beu Arg Ser Glu Asp ThrAla Phe Tyr Phe Cys Ala Ser FR4: Trp Gly Gln Gly Thr Thr Val Thr Val SorSer
 19. The H chain according to claim 15 wherein said human C region isa γ-1C region or γ-4C region.
 20. An antibody to human medulloblastomacells composed of the L chain as set forth in claim 2 and the H chain asset forth in claim
 12. 21. An antibody according to claim 20 wherein theFRs of said V region are derived from a mouse antibody.
 22. The antibodyas set forth in claim 20 wherein the FRs of said V region are derivedfrom a human antibody.
 23. A DNA coding for an L chain of an antibody tohuman medulloblastoma cells comprising an L chain V region containingthree CDRs having the amino acids as set forth in claim 1 or a portionthereof and four FRs, and a human L chain C region.
 24. A DNA accordingto claim 23 wherein said L chain V region has a nucleotide sequenceindicated in SEQ ID NO:58, 61, 63, 66, 70 or
 73. 25. A DNA coding for anH chain of an antibody to human medulloblastoma cells comprising an Hchain V region containing three CDRs having the amino acids as set forthin claim 11 or a portion thereof and four FRs, and a human H chain Cregion.
 26. A DNA according to claim 25 wherein said H chain V regionhas the nucleotide sequence indicated in SEQ ID NO:80.
 27. A recombinantvector comprising a DNA according to claim 23 or 24 or a portionthereof.
 28. A recombinant vector comprising a DNA according to claim 25or 26 or a portion thereof.
 29. A transformant co-transformed with arecombinant vector according to claim 27 and the recombinant vector asset forth in claim
 28. 30. A process for producing antibody to humanmedulloblastoma cells using gene recombination technology comprising aculturing a transformant according to claim 29 and then isolating atarget antibody produced.
 31. A single-chain Fv composed by linking an Hchain V region according to claim 11 with an L chain V region as setforth in claim I by means of a peptide linker.
 32. A single-chain Fvaccording to claim 31 wherein said linker peptide has the followingamino acid sequence: Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly GlyGly Ser
 33. A single-chain Fv according to claim 31 or 32 comprising anH chain V region having an amino acid sequence of amino acid numbersfrom 1 to 116 in the amino acid sequence set forth in SEQ ID NO:80, andan L chain V region having an amino acid sequence of amino acid numbersfrom 1 to 106 in the amino acid sequence set forth in SEQ ID NO:40, 43,46, 47, 50, 51, 54, 55, 58, 61,
 62. 63, 66, 69, 70 or
 73. 34. Asingle-chain Pv according to claim 31 or 32 comprising an H chain Vregion having an amino acid sequence of amino acid numbers from 1 to 116i:: the amino acid sequence set forth in SEQ ID NO. 80, and an L chain Vregion having an amino acid sequence of amino acid numbers from 1 to 106in the amino acid sequence set forth in SEQ ID NO:73.
 35. A single-chainFv according to claim 31 having an amino acid sequence as set forth inSEQ ID NO;
 39. 36. A DNA coding for a single-chain Fv according to anyone of claims 31 to
 35. 37. A recombinant vector comprising a DNAaccording to claim
 36. 38. A host transfected with a recombinant vectoraccording to claim
 37. 39. A process for producing a single-chain Fvcomprising culturing a transformant according To claim 38 and recoveringsingle-chain Fv region from said culture.